Methods for modifying the production of a polypeptide

ABSTRACT

The present invention relates to methods for modifying the production of a polypeptide, comprising: (a) introducing a nucleic acid construct into a cell, wherein the cell comprises a DNA sequence encoding a polypeptide, under conditions in which the nucleic acid construct integrates into the genome of the cell at a locus not within the DNA sequence encoding the polypeptide to produce a mutant cell, wherein the integration of the nucleic acid construct modifies the production of the polypeptide by the mutant cell relative to the cell when the mutant cell and the cell are cultured under the same conditions; and (b) identifying the mutant cell with the modified production of the polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No. 8/713,312 filed on Sep. 13, 1996, now abandoned, which application is fully incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods for modifying the production of a polypeptide by a cell.

2. Description of the Related Art

Several methods have been used to modify the production of polypeptides by mutagenizing cells. For example, the production of proteins has been altered by producing mutant cells by classical mutagenesis which involves treating cells with chemical, physical, and biological agents as mutagenic (mutation inducing) agents to increase the frequency of mutational events.

Production of proteins also has been modified by mutagenesis of a cell with short sections of double-stranded DNA, consisting of more than 2000 base pairs, called transposons which usually code for resistance to one or sometimes several antibiotics. Transposons are able to move or jump within the genome, even between a bacterial chromosome and a plasmid, and they are able to become integrated in a number of different sites on the genome. An insertion of a transposon within a structural gene interrupts the normal nucleotide sequence of the gene so that it can no longer deliver the information for the synthesis of the normal, functional polypeptide (Seifert et al., 1986, Proceedings of the National Academy of Sciences USA 83: 735-739). An insertion also may disrupt a gene whose gene product is required for expression (Marquez-Magana and Chamberlin, 1994, Journal of Bacteriology 176: 2427-2434). In addition, Errede et al. (1980, Cell 22: 427-436) disclose the insertion of a transposable element adjacent to the structural gene coding for iso-2-cytochrome c causing overproduction. Furthermore, WO 96/29414 discloses that transposable elements may be constructed containing a transposon and a DNA sequence capable of regulating a targeted gene where upon introduction into a cell the transposable element integrates into the genome of the cell in a manner which regulates the expression of the gene.

A widely used method for increasing production of a polypeptide is amplification to produce multiple copies of the gene encoding the polypeptide. For example, U.S. Pat. No. 5,578,461 discloses the inclusion via homologous recombination of an amplifiable selectable marker gene in tandem with the gene where cells containing amplified copies of the selectable marker can be selected for by culturing the cells in the presence of the appropriate selectable agent.

In addition, the production of polypeptides has been increased by replacing one promoter with a different promoter or one signal peptide coding region with another. See, e.g., U.S. Pat. No. 5,641,670.

Methods for altering gene expression by disrupting genes encoding various regulatory elements have also been described. For example, Toma et al. (1986, Journal of Bacteriology 167: 740-743) showed that a deletion from -156 to -90 in the npr promoter region caused overexpression of the neutral protease encoded by the npr gene. Pero and Sloma (1993, In A. L. Sonensheim, J. A. Hoch, and R. Losick, editors, Bacillus subtilis and Other Gram-Positive Bacteria, pp. 939-952, American Society for Microbiology, Washington, D.C.) disclose that mutating the sporulation gene spoOA results in deficient synthesis of proteases and that mutations in the abrB gene restore synthesis.

The production of polypeptides also has been increased by disrupting DNA sequences encoding a protease capable of hydrolyzing the polypeptide under the conditions for producing the polypeptide.

The secretion of polypeptides has also been modified by overproduction of secretion proteins (Ruohonen et al., 1997, Yeast, 13: 337-351), and producing a super-secreting cell (U.S. Pat. No. 5,312,735).

Methods for increasing the production of metabolites have also been described. For example, WO 96/41886 discloses that increased production of clavam produced by an organism having at least part of the clavam pathway and at least part of a cephalosporin pathway by interfering with the conversion of L-lysine to L-alpha-aminoadipic acid in the cephalosporin pathway. WO 94/13813 discloses the disruption of gene which encodes a protein which degrades betaine, an enzyme inducer.

It is an object of the present invention to provide new and improved methodologies for altering production of polypeptides and metabolites.

BRIEF SUMMARY OF THE INVENTION

The present invention is drawn to methods for modifying the production of a polypeptide by a cell. In the methods of the present invention, a nucleic acid construct is introduced into a cell which contains a DNA sequence encoding a specific polypeptide. The introduced nucleic acid construct integrates into the host genome at a locus not within the DNA sequence of interest to produce a mutant cell. The integration of the nucleic acid construct into the locus modifies the production of the polypeptide by the mutant cell relative to the parent cell. Mutant cells are then identified in which the polypeptide's production is modified by the mutant cell relative to the parent cell. Modification is determined by comparing production of the polypeptide when the mutant cell and the parent cell are cultured under the same conditions.

An advantage of the present invention is that the mutation can be recovered and leads to a modification of the production of a polypeptide encoded by a DNA sequence which does not contain the mutation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a restriction map of pJaL292.

FIG. 2 is a restriction map of pKS6.

FIG. 3 is a restriction map of pBANe13.

FIG. 4 is a restriction map of pBANe6.

FIG. 5 is a restriction map of pMHan37.

FIG. 6 is a restriction map of pBANe8.

FIG. 7 is a restriction map of pSO2.

FIG. 8 is a restriction map of pSO122 and shows the construction of pDSY81 and pDSY82 from pSO122.

FIG. 9 is the nucleic acid sequence and the deduced amino acid sequence of the rescued locus of mutant Aspergillus oryzae DEBY599.3 (SEQ ID NOS:9 and 10, respectively).

FIG. 10 is the nucleic acid sequence and the deduced amino acid sequence of the rescued locus of mutant Aspergillus oryzae DEBY10.3 (SEQ ID NOS:16 and 17).

FIG. 11 is a restriction map of pJaL400.

FIG. 12 is the construction of pMT1935.

FIG. 13 is a restriction map of pJaL394.

FIG. 14 is a restriction map of pMT1931.

FIG. 15 is a restriction map of pMT1936.

FIG. 16 is the nucleic acid sequence and the deduced amino acid sequence of the rescued locus of mutant Aspergillus oryzae DEBY932 (SEQ ID NOS:25 and 26).

FIG. 17 is the nucleic acid sequence and deduced amino acid sequence of the rescued locus of mutant Aspergillus oryzae DEBY1058 (SEQ ID NOS:29 and 30).

FIG. 18 is a restriction map of pDSY161.

FIG. 19 is a restriction map of pDSY162.

FIG. 20 is the nucleic acid sequence of the rescued locus of mutant Aspergillus oryzae 1204.3.3 (SEQ ID NO:34).

FIG. 21 is the nucleic acid sequence of the rescued locus of mutant Aspergillus oryzae H603 (SEQ ID NO:39).

FIG. 22 is a restriction map of pGAG3.

FIG. 23 is a restriction map of pJaL389.

FIG. 24 is a restriction map of pJaL335.

FIG. 25 is a restriction map of pJaL399.

FIG. 26 is a restriction map of pDM176.

FIG. 27 is a restriction map of pHB218.

FIG. 28 is a restriction map of pSE39.

FIG. 29 is a restriction map of pDSY153.

FIG. 30 is a restriction map of pCaHj505.

FIG. 31 is a restriction map of pMStr107.

FIG. 32 is the nucleic acid sequence and deduced amino acid sequence of the rescued locus of mutant Aspergillus oryzae P4-8.1 (SEQ ID NOS:50 and 51).

FIG. 33 is the nucleic acid sequence and deduced amino acid sequence of the rescued locus of mutant Aspergillus oryzae P7-14. 1 (SEQ ID NOS:56 and 57).

FIG. 34 is a restriction map of pMT1612.

FIG. 35 is the nucleic acid sequence and deduced amino acid sequence of the rescued locus of mutant Aspergillus oryzae DEBY7-17.2 (SEQ ID NOS:63 and 64).

FIG. 36 is the nucleic acid sequence of the rescued locus of mutant Aspergillus oryzae DEBY3-2.1 (SEQ ID NO:66).

FIG. 37 is the nucleic acid sequence of the rescued locus of mutant Aspergillus oryzae DEBY5-7.1 (SEQ ID NO:71).

FIG. 38 is the nucleic acid sequence of the rescued locus of mutant Aspergillus oryzae DEBY8-0.1 (SEQ ID NO:76).

DETAILED DESCRIPTION OF THE INVENTION

In a first embodiment, the present invention relates to methods of producing a polypeptide, comprising

(a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein

(i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, not within a second DNA sequence encoding a protein that negatively regulates transcription, translation or secretion of the polypeptide, and not within a third DNA sequence encoding a protease capable of hydrolyzing the polypeptide under the conditions; and

(ii) the mutant cell produces more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and

(b) recovering the polypeptide.

A "protein that negatively regulates transcription" is defined herein as a repressor that negatively affects the process of RNA synthesis by RNA polymerase to produce a single-stranded RNA complementary to a DNA sequence, or as a protein that degrades an enzyme inducer which is generally a chemical agent produced by a biosynthetic or catabolic pathway of a cell. The repressor consists of distinct domains that are required for DNA-binding, transcription repression, and inducer or repressor binding.

A "protein that negatively regulates translation" is defined herein as a protein or a substance, the production of which is catalyzed by the protein, that negatively affects the process of protein synthesis carried out by ribosomes which de-code the information contained in mRNA derived from transcription of a gene. For example, the substance may be a cap-dependent translation initiation factor, e.g., p20 (Altmann et al., 1997, EMBO Journal 16: 1114-1121); or a sex-lethal protein, e.g., the sex-lethal protein of Drosophila which regulates the translation of ms1-2 (Bashaw and Baker, 1997, Cell 89: 789-798).

A "protein that negatively regulates secretion" is defined herein as a protein or a substance, the production of which is catalyzed by the protein, that negatively affects the process of transferring a protein molecule through a membrane into (i) an intracellular compartment, e.g., a vacuole or mitochrondrion, (ii) the periplasmic space, or (iii) the culture medium and, in eukaryotic cells, the process of vesicular transport that ultimately results in exocytic release of secreted proteins from the cell. The secretory process oversees and promotes correct protein folding, mediates any required post-translational modifications (such as glycosylation), and sorts, processes, and targets proteins to specific cellular sites all at a rate consistent with the function of the cell as a whole. Such substances include a protein with Ca²⁺ -ATPase activity which upon inactivation increase levels of secreted heterologous or mutant proteins (for example, see Rudolph et al., 1989, Cell 58: 133-145); or the binding protein BiP which is an ATP-dependent hsp70-class chaperone found in the endoplasmic reticulum of eukaryotic cells which when decreased in mammalian cells through the use of anti-sense RNA results in up to a three-fold increase in secreted levels of a mutant protein (Dorner et al., 1988, Molecular and Cellular Biology 8: 4063-4070). In a specific embodiment, the substance is a protein with ATPase activity or the binding protein BiP.

In a second embodiment, the present invention relates to methods of producing a polypeptide, comprising

(A) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein

(i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, wherein the introduction of the nucleic acid construct disrupts a gene encoding an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, or regulatory or control sequences thereof, other than a gene encoding a protease which is capable of hydrolyzing the polypeptide under the conditions; and

(ii) the mutant cell produces more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and

(B) recovering the polypeptide.

A mutant cell that "produces" more of a polypeptide is defined herein as a cell from which more of the polypeptide is recovered relative to the parent cell.

In a third embodiment, the present invention relates to methods of producing a polypeptide, comprising

(a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein

(i) the mutant cell is related to a parent cell; which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, not within a second DNA sequence encoding a protein that negatively regulates transcription of the polypeptide, and not within a third DNA sequence encoding a protease capable of hydrolyzing the polypeptide under the conditions; and

(ii) the mutant cell expresses more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and

(b) recovering the polypeptide.

In a fourth embodiment, the present invention relates to methods of producing a polypeptide, comprising

(A) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein

(i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, wherein the introduction of the nucleic acid construct disrupts a gene encoding an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, or regulatory or control sequences thereof, other than a gene encoding a protease which is capable of hydrolyzing the polypeptide under the conditions; and

(ii) the mutant cell expresses more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and

(B) recovering the polypeptide.

A mutant cell that "expresses" more of a polypeptide is defined herein as a cell that contains an increase in functional mRNA encoding the polypeptide relative to the parent cell. It will be understood that an increase in functional mRNA may result from an increase in the absolute rate of transcription of the gene encoding the polypeptide and/or from alterations in post-transcriptional processing or modification of the transcripts, including nuclear-cytoplasmic transport and/or cytoplasmic stabilization of the mRNA. Such mutant cells may be identified using conventional techniques, including without limitation Northern blot analysis, run-off transcription assays, and the like.

In a fifth embodiment, the present invention relates to methods of producing a polypeptide, comprising

(a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein

(i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, not within a second DNA sequence encoding a protein that negatively regulates translation of the polypeptide, and not within a third DNA sequence encoding a protease capable of hydrolyzing the polypeptide under the conditions; and

(ii) the mutant cell synthesizes more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and

(b) recovering the polypeptide.

In a sixth embodiment, the present invention relates to methods of producing a polypeptide, comprising

(A) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein

(i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, wherein the introduction of the nucleic acid construct disrupts a gene encoding an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, or regulatory or control sequences thereof, other than a gene encoding a protease which is capable of hydrolyzing the polypeptide under the conditions; and

(ii) the mutant cell synthesizes more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and

(B) recovering the polypeptide.

A mutant cell that "synthesizes" more of a polypeptide is defined herein as a cell that accumulates larger amounts of the polypeptide relative to a parent cell. Accumulation refers to the total amount of the polypeptide in the culture as a whole, i.e., in both intracellular and extracellular compartments taken together. Such mutant cells may be identified using any suitable technique, including without limitation pulse-labelling or steady-state labelling using radiolabelled amino acids; immunoblot analysis of cell and medium fractions using an antibody specific to the polypeptide; assays of biological activity; separation by conventional chromatographic methods; and the like.

In a seventh embodiment, the present invention relates to methods of producing a polypeptide, comprising

(a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein

(i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, not within a second DNA sequence encoding a protein that negatively regulates secretion of the polypeptide, and not within a third DNA sequence encoding a protease capable of hydrolyzing the polypeptide under the conditions; and

(ii) the mutant cell secretes more of the polypeptide than the parent cell when both cells are cultivated under the conditions;

(b) recovering the polypeptide.

In an eighth embodiment, the present invention relates to methods of producing a polypeptide, comprising

(A) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein

(i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, wherein the introduction of the nucleic acid construct disrupts a gene encoding an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, or regulatory or control sequences thereof, other than a gene encoding a protease which is capable of hydrolyzing the polypeptide under the conditions; and

(ii) the mutant cell secretes more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and

(B) recovering the polypeptide.

A mutant cell that "secretes" more of a polypeptide is defined herein as a cell in which the amount of the polypeptide released into the extracellular medium is increased relative to the parent cell. Such mutant cells may be identified using, e.g., pulse-chase labelling in conjunction with immunoprecipitation to quantify the proportion of the newly synthesized polypeptide that is externalized as well as the absolute amount released in the mutant cell relative to the parent cell. Immunoblot analysis, biological activity assays, and physical-chemical separation methods may also be used to quantify the absolute amounts of the polypeptide released in mutant vs. parent cells.

In a ninth embodiment, the present invention relates to methods of producing a polypeptide, comprising

(a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein

(i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the random integration of a nucleic acid construct into the genome of the parent cell at a locus wherein the nucleic acid construct is not homologous with the locus and wherein the locus is not within the first DNA sequence nor within a second DNA sequence encoding a protease capable of hydrolyzing the polypeptide under the conditions; and

(ii) the mutant cell produces more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and

(b) recovering the polypeptide.

In a tenth embodiment, the present invention relates to methods of producing a polypeptide, comprising

(a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein

(i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence and a second DNA sequence encoding a protein that positively regulates transcription, translation or secretion of the polypeptide; and

(ii) the mutant cell produces less of the polypeptide than the parent cell when both cells are cultivated under the conditions; and

(b) recovering the polypeptide.

A mutant cell that "produces" less which less of defined herein as a cell from which less of the polypeptide is recovered relative to the parent cell.

A "protein that positively regulates transcription" is defined herein as an activator or an inducer that positively affects the process of RNA synthesis by RNA polymerase to produce a single-stranded RNA complementary to a DNA sequence. The activator consists of distinct domains that are required for DNA-binding, transcription activation, and inducer or repressor binding. An inducer is generally a chemical agent produced by a biosynthetic or catabolic pathway of a cell. In a specific embodiment, the substance is an activator or an inducer.

A "protein that positively regulates translation" is defined herein as a protein or a substance, the production of which is catalyzed by the protein, that positively affects the process of protein synthesis carried out by ribosomes which de-code the information contained in mRNA derived from transcription of a gene. In a specific embodiment, the substance is an initiation factor or an elongation factor.

A "protein that positively affects secretion" is defined herein as a protein or a substance, the production of which is catalyzed by the protein, that positively affects the process of transferring a protein molecule through a membrane into (i) an intracellular compartment, e.g., a vacuole or mitochrondrion, (ii) the periplasmic space, or (iii) the culture medium or positively affects vesicular transport as described above. Such substances include folding proteins, e.g., protein disulfide isomerase and peptidyl prolyl isomerase isoforms; chaperones, e.g., heat shock proteins, signal recognition particles, PrsA, SecD, SecF, and BiP; translocating chain-associating membrane proteins (TRAM); translocase complexes; and processing enzymes, e.g., glycosylating enzymes; signal peptidases; pro region peptidases. In a specific embodiment, the substance is a folding protein, a chaperone, a signal recognition particle, PrsA, SecD, SecF, BiP, a translocating chain-associating membrane, a translocase complex, or a processing enzyme.

Other embodiments of the present invention relate to methods for producing polypeptides as described in the tenth embodiment, except that the mutant cells express, synthesize or secrete less of the polypeptide than the parent cell when both cells are cultivated under the conditions.

A mutant cell that "expresses" less of a polypeptide is defined herein as a cell that contains a decrease in functional mRNA encoding the polypeptide relative to the parent cell. It will be understood that a decrease in functional mRNA may result from a decrease in the absolute rate of transcription of the gene encoding the polypeptide and/or from alterations in post-transcriptional processing or modification of the transcripts, including nuclear-cytoplasmic transport and/or cytomplasmic stabilization of the mRNA. Such mutant cells may be identified using conventional techniques, including without limitation Northern blot analysis, run-off transcription assays, and the like.

A mutant cell that "synthesizes" less of a polypeptide is defined herein as a cell that accumulates smaller amounts of the polypeptide relative to a parent cell. Accumulation refers to the total amount of the polypeptide in the culture as a whole, i.e., in both intracellular and extracellular compartments taken together. Such mutant cells may be identified using any suitable technique, including without limitation pulse-labelling or steady-state labelling using radiolabelled amino acids; immunoblot analysis of cell and medium fractions using an antibody specific to the polypeptide; assays of biological activity; separation by conventional chromatographic methods; and the like.

A mutant cell that "secretes" less of a polypeptide is defined herein as a cell in which the amount of the polypeptide released into the extracellular medium is decreased relative to the parent cell. Such mutant cells may be identified using, e.g., pulse-chase labelling in conjunction with immunoprecipitation to quantify the proportion of the newly synthesized polypeptide that is externalized as well as the absolute amount released in the mutant cell relative to the parent cell. Immunoblot analysis, biological activity assays, and physical-chemical separation methods may also be used to quantify the absolute amounts of the polypeptide released in mutant vs. parent cells.

The present invention also relates to methods of producing a metabolite, comprising

(A) cultivating a mutant cell under conditions conducive for production of the metabolite, wherein

(i) the mutant cell is related to a parent cell, which comprises one or more first DNA sequences encoding first polypeptides in the biosynthetic pathway of the metabolite, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within (a) the first DNA sequences, (b) a second DNA sequence encoding a substance that negatively regulates transcription, translation or secretion of the polypeptides, (c) a third DNA sequence encoding a protease capable of hydrolyzing any of the first polypeptides under the conditions, and (d) one or more fourth DNA sequences encoding a second polypeptide in the second biosynthetic pathway of a second metabolite wherein the biosynthetic pathway and the second biosynthetic pathway involve the production of the same intermediate and the second polypeptide catalyzes a step after the production of the intermediate; and

(ii) the mutant cell produces more of the metabolite than the parent cell when both cells are cultivated under the conditions; and

(B) recovering the metabolite.

The present invention also relates to methods of producing a metabolite, comprising

(A) cultivating a mutant cell under conditions conducive for production of the metabolite, wherein

(i) the mutant cell is related to a parent cell, which comprises one or more first DNA sequences encoding first polypeptides in the biosynthetic pathway of the metabolite, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within (a) the first DNA sequences, (b) a second DNA sequence encoding a protein that negatively regulates transcription, translation or secretion of the polypeptides, and (c) one or more third DNA sequences encoding a second polypeptide in the second biosynthetic pathway of a second metabolite wherein the biosynthetic pathway and the second biosynthetic pathway involve the production of the same intermediate and the second polypeptide catalyzes a step prior to the production of the intermediate; and

(ii) the mutant cell produces less of the metabolite than the parent cell when both cells are cultivated under the conditions; and

(B) recovering the metabolite.

The present invention also relates to methods of producing a first polypeptide, comprising

(a) forming a mutant cell by introducing a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, a second DNA sequence encoding a protein that negatively regulates transcription, translation or secretion of a second polypeptide, and a third DNA sequence encoding a protease capable of hydrolyzing the polypeptide under conditions conducive to the production of the first polypeptide;

(b) isolating the mutant cell which produces more of the polypeptide than the parent cell when both cells are cultivated under the conditions;

(c) identifying the locus wherein the nucleic acid construct has been integrated;

(d) producing a cell in which a corresponding locus has been disrupted;

(e) culturing the cell under the conditions conducive; and

(f) recovering the first polypeptide.

A corresponding locus is defined herein as a locus which encodes a polypeptide with has the same function as the polypeptide encoded by the rescued locus.

The present invention also relates to methods of producing a polypeptide, comprising

(a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein

(i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence and a second DNA sequence encoding a protease capable of hydrolyzing the polypeptide under the conditions, wherein the introduction of the nucleic acid construct specifically enhances transcription, translation or secretion of the polypeptide; and

(ii) the mutant cell produces more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and

(b) recovering the polypeptide.

"Specific" enhancement of transcription, translation, or secretion as used herein refers to an enhancement of one or more aspects of the biogenesis and production of the polypeptide that is limited to the polypeptide of interest and is not accompanied by a global effect on other polypeptides in the cell. Preferably, specific enhancement affects only a small number of polypeptides, including the polypeptide of interest. Most preferably, specific enhancement affects only the polypeptide of interest.

Global enhancement of these biogenetic processes can be distinguished from specific enhancement using conventional methods that are well known in the art. For example, biosynthetic pulse-labelling with ³ H-uridine (followed by quantitation of total radioactivity incorporated into RNA) can be used to determine that a mutant cell does not generally synthesize mRNA at a higher or lower rate than the parent cell to which it is related. Similarly, pulse-labelling with, e.g., ³⁵ S-methionine (followed by quantitation of total radioactivity incorporated into protein) can be used to determine that a mutant cell does not generally synthesize proteins at a higher or lower rate than the parent cell to which it is relate. General rates of secretion can be compared between mutant and parent cells by pulse-chase labelling using radioactive amino acids or sugars followed by quantitation of extracellular vs. intracellular radioactivity.

These methods can also be used to determine if transcription, translation, or secretion of a limited number of other polypeptides might also be affected in the mutant cell. For example, capture of specific radiolabelled RNA transcripts by hybridization to an immobilized oligonucleotide probe can be used to assess transcription rates of individual genes. For translation and secretion, resolution of radiolabelled nascent proteins by, e.g., SDS-PAGE (with or without immunoprecipitation of individual proteins) can be used to compare instantaneous rates of translation and/or secretion of individual proteins.

Polypeptides

The term "polypeptide" encompasses peptides, oligopeptides, and proteins and, therefore, is not limited to a specific length of the encoded product. The polypeptide may be native to the cell or may be a heterologous polypeptide. Preferably, it is a heterologous polypeptide. The polypeptide may also be a recombinant polypeptide which is a polypeptide native to a cell, which is encoded by a nucleic acid sequence which comprises one or more control sequences foreign to the gene. The polypeptide may be a wild-type polypeptide or a variant thereof. The polypeptide may also be a hybrid polypeptide which contains a combination of partial or complete polypeptide sequences obtained from at least two different polypeptides where one or more of the polypeptides may be heterologous to the cell. Polypeptides further include naturally occurring allelic and engineered variations of the above mentioned polypeptides.

In a preferred embodiment, the polypeptide is an antibody or portions thereof.

In a preferred embodiment, the polypeptide is an antigen.

In a preferred embodiment, the polypeptide is a clotting factor.

In a preferred embodiment, the polypeptide is an enzyme.

In a preferred embodiment, the polypeptide is a hormone or a hormone variant.

In a preferred embodiment, the polypeptide is a receptor or portions thereof.

In a preferred embodiment, the polypeptide is a regulatory protein.

In a preferred embodiment, the polypeptide is a structural protein.

In a preferred embodiment, the polypeptide is a reporter.

In a preferred embodiment, the polypeptide is a transport protein.

In a more preferred embodiment, the polypeptide is an oxidoreductase,

In a more preferred embodiment, the polypeptide is a transferase.

In a more preferred embodiment, the polypeptide is a hydrolase.

In a more preferred embodiment, the polypeptide is a lyase.

In a more preferred embodiment, the polypeptide is an isomerase.

In a more preferred embodiment, the polypeptide is a ligase.

In an even more preferred embodiment, the polypeptide is an aminopeptidase

In an even more preferred embodiment, the polypeptide is an amylase.

In an even more preferred embodiment, the polypeptide is a carbohydrase.

In an even more preferred embodiment, the polypeptide is a carboxypeptidase.

In an even more preferred embodiment, the polypeptide is a catalase.

In an even more preferred embodiment, the polypeptide is a cellulase.

In an even more preferred embodiment, the polypeptide is a chitinase.

In an even more preferred embodiment, the polypeptide is a cutinase.

In an even more preferred embodiment, the polypeptide is a deoxyribonuclease.

In an even more preferred embodiment, the polypeptide is a dextranase.

In an even more preferred embodiment, the polypeptide is an esterase.

In an even more preferred embodiment, the polypeptide is an alpha-galactosidase.

In an even more preferred embodiment, the polypeptide is a beta-galactosidase.

In an even more preferred embodiment, the polypeptide is a glucoamylase.

In an even more preferred embodiment, the polypeptide is an alpha-glucosidase.

In an even more preferred embodiment, the polypeptide is a beta-glucosidase.

In an even more preferred embodiment, the polypeptide is a haloperoxidase.

In an even more preferred embodiment, the polypeptide is an invertase.

In an even more preferred embodiment, the polypeptide is a laccase.

In an even more preferred embodiment, the polypeptide is a lipase.

In an even more preferred embodiment, the polypeptide is a mannosidase.

In an even more preferred embodiment, the polypeptide is a mutanase.

In an even more preferred embodiment, the polypeptide is an oxidase.

In an even more preferred embodiment, the polypeptide is a pectinolytic enzyme.

In an even more preferred embodiment, the polypeptide is a peroxidase.

In an even more preferred embodiment, the polypeptide is a phytase.

In an even more preferred embodiment, the polypeptide is a polyphenoloxidase.

In an even more preferred embodiment, the polypeptide is a proteolytic enzyme.

In an even more preferred embodiment, the polypeptide is a ribonuclease.

In an even more preferred embodiment, the polypeptide is a transglutaminase.

In an even more preferred embodiment, the polypeptide is a xylanase.

In an even more preferred embodiment, the polypeptide is human insulin or an analog thereof.

In an even more preferred embodiment, the polypeptide is human growth hormone.

In an even more preferred embodiment, the polypeptide is erythropoietin.

In an even more preferred embodiment, the polypeptide is insulinotropin.

The polypeptide also may be an enzyme involved in the biosynthesis of a specific metabolite. The biosynthesis of a metabolite generally involves a biosynthetic pathway containing an array of enzyme-catalyzed chemical reaction steps in which one or more steps may be rate-limiting. In this embodiment of the present invention, the integration of the nucleic acid construct into the cell's genome modifies the production of the metabolite by modifying one or more of these enzyme-catalyzed steps.

The metabolite may be any organic compound of a cell which has been produced by transformation of a precursor organic compound by an enzyme-catalyzed chemical reaction of the cell. The metabolite may be a primary metabolite or a secondary metabolite. Furthermore, the metabolite may be a biosynthetic pathway intermediate or a biosynthetic pathway product. Preferably, the metabolite is an alkaloid, an amino acid, an antibiotic, a cofactor, a drug, a fatty acid, a fungicide, a herbicide, an insecticide, an organic acid, a prosthetic group, a rodenticide, a sweetener, a vitamin, a deoxysugar, a surfactant, a mycotoxin, an organic acid, a sugar alcohol, a toxic metabolite, or a toxin.

Nucleic Acid Constructs

The nucleic constructs used in the methods of the present invention may be termed "tagged nucleic acid constructs". "A tagged nucleic acid construct" is a nucleic acid molecule containing an identifiable nucleic acid sequence which integrates into the cell's genome at one or more loci thereby marking the loci. The genome is the complete set of DNA of a cell including chromosomal and artificial chromosomal DNA and extrachromosomal DNA, i.e., self-replicative genetic elements.

The nucleic acid constructs may be any nucleic acid molecule, either single- or double-stranded, which is synthetic DNA, isolated from a naturally occurring gene, or has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature. The nucleic acid constructs may be circular or linear. Furthermore, the nucleic acid constructs may be contained in a vector, may be a restriction enzyme cleaved linearized fragment, or may be a PCR amplified linear fragment.

The nucleic acid constructs may contain any nucleic acid sequence of any size. In one embodiment, the nucleic acid constructs are between about 10-20,000 bp in length, preferably 100-15,000 bp in length, more preferably 500-15,000 bp in length, even more preferably 1000-15,000 bp in length, and most preferably 1,000-10,000 bp in length.

Preferably, the nucleic acid constructs have less than 40% homology, preferably less than 30% homology, more preferably less than 20% homology, even more preferably less than 10% homology, and most preferably no homology with the locus.

Preferably, the nucleic acid constructs have less than 40% homology, preferably less than 30% identity, more preferably less than 20% identity, even more preferably less than 10%, and most preferably no homology with the DNA sequence encoding the polypeptide of interest.

The nucleic acid construct can be introduced into a cell as two or more separate fragments. In the event two fragments are used, the two fragments share DNA sequence homology (overlap) at the 3' end of one fragment and the 5' end of the other. Upon introduction into a cell, the two fragments can undergo homologous recombination to form a single fragment. The product fragment is then in a form suitable for recombination with the cellular sequences. More than two fragments can be used, designed such that they will undergo homologous recombination with each other to ultimately form a product suitable for recombination with a cellular sequence.

It will be further understood that two or more nucleic acid constructs may be introduced into the cell as circular or linear fragments using the methods of the present invention, wherein the fragments do not contain overlapping regions as described above. It is well known in the art that for some organisms, the introduction of multiple constructs into a cell results in their integration at the same locus.

The nucleic acid constructs can contain coding or non-coding DNA sequences. Coding sequences are sequences which are capable of being transcribed into mRNA and translated into a polypeptide when placed under the control of the appropriate control sequences. The boundaries of a coding sequence are generally determined by a translation start codon ATG at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

In a preferred embodiment, the nucleic acid constructs contain a selectable marker as the identifiable nucleic acid sequence. A selectable marker is a gene, the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Integration of a selectable marker into the genome of a host cell permits easy selection of transformed cells. Selectable marker genes for use in the methods of the present invention include, but are not limited to, acetamidase (amdS), 5-aminolevulinic acid synthase (hemA), anthranilate synthase (trpC), glufosinate resistance genes, hygromycin phosphotransferase (hygB), nitrate reductase (niaD), ornithine carbamoyltransferase (argB), orotidine-5'-phosphate decarboxylase (pyrG), phosphinothricin acetyltransferase (bar), and sulfate adenyltransferase (sC), as well as equivalents from other species. In a more preferred embodiment, the selectable marker is the amdS gene of Aspergillus nidulans or Aspergillus oryzae, the bar gene of Streptomyces hygroscopicus, the hemA gene of Aspergillus oryzae or the pyrG gene of Aspergillus nidulans or Aspergillus oryzae. Other selectable markers for use in the methods of the present invention are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers which confer antibiotic resistance such as ampicillin (amp), kanamycin (kan), chloramphenicol (cam) or tetracycline resistance (tet). A frequently used mammalian marker is the dihydrofolate reductase gene (dfhr). Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

In another preferred embodiment, the constructs comprise vector sequences alone or in combination with a selectable marker, including vector sequences containing an origin of replication, e.g., E. coli vector sequences such as pUC19, pBR322, or pBluescript. For example, an E. coli vector sequence containing an origin of replication can facilitate recovery of the construct from the host genome after integration due to the E. coli origin of replication. The construct can be recovered from the host genome by digestion of the genomic DNA with a restriction endonuclease followed by ligation of the recovered construct and transformation of E. coli.

In a preferred embodiment, the nucleic acid constructs do not contain the coding sequence of the DNA sequence for the polypeptide or portions thereof. In another preferred embodiment, the nucleic acid constructs contain a sequence which is not homologous to the DNA sequence encoding the polypeptide in order to block the construct from integrating or disrupting the DNA sequence of interest.

In another preferred embodiment, the nucleic acid constructs contain one or more copies of the DNA sequence coding for the polypeptide operably linked to control sequences. In this embodiment, the production of the polypeptide will be modified by both gene inactivation and the introduction of one or more copies of the DNA sequence.

In another preferred embodiment, the nucleic acid constructs do not contain transposable elements, i.e., transposons. A transposon is a discrete piece of DNA which can insert itself into many different sites in other DNA sequences within the same cell. The proteins necessary for the transposition process are encoded within the transposon. A copy of the transposon may be retained at the original site after transposition. The ends of a transposon are usually identical but in inverse orientation with respect to one another.

In another preferred embodiment, the nucleic acid constructs may contain one or more control sequences, e.g., a promoter alone or in combination with a selectable marker, wherein the control sequences upon integration are not operably linked to the DNA sequence encoding the polypeptide of interest. The term "operably linked" is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the production of a primary RNA transcript. Such control sequences are a promoter, a signal sequence, a propeptide sequence, a transcription terminator, a polyadenylation sequence, an enhancer sequence, an attenuator sequence, and an intron splice site sequence. Each control sequence may be native or foreign to the cell or to the polypeptide-coding sequence.

The presence of a strong promoter in the nucleic acid construct allows for additional genetic effects in addition to gene inactivation via insertion into a structural gene (or functional transcriptional promoter or mRNA termination regions). The promoter may insert upstream of a structural gene so as to enhance its transcription. Alternatively, if the promoter sequences insert in reverse gene orientation so as to generate antisense RNA, there is the possibility of gene inactivation in diploid or higher ploidy cells. By the same mechanism, insertion of the promoter sequences in reverse orientation may result in inactivation of multiple gene family encoded gene product activities.

In another preferred embodiment, the nucleic acid constructs contain a control sequence other than a promoter.

In another preferred embodiment, the nucleic acid constructs do not contain control sequences.

Locus

In the methods of the present invention, the nucleic acid constructs are introduced at a "locus not within the DNA sequence of interest" or a "locus not within DNA sequences encoding polypeptides in the biosynthetic pathway of a metabolite" which means that the nucleic acid construct is not introduced into the polypeptide-coding sequence, the control sequences thereof, and any intron sequences within the coding sequence.

Control sequences include all components which are operably linked to the DNA sequence and involved in the expression of the polypeptide-coding sequence. Such control sequences are a promoter, a signal sequence, a propeptide sequence, a transcription terminator, a polyadenylation sequence, an enhancer sequence, an attenuator sequence, and an intron splice site sequence. Each of the control sequences may be native or foreign to the coding sequence.

The promoter sequence contains transcriptional control sequences which mediate the expression of the polypeptide. The promoter may be any promoter sequence including mutant, truncated, and hybrid promoters.

The signal peptide coding region codes for an amino acid sequence linked to the amino terminus of the polypeptide which can direct the expressed polypeptide into the cell's secretory pathway.

The propeptide coding region codes for an amino acid sequence positioned at the amino terminus of the polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.

The terminator is a sequence operably linked to the 3' terminus of the polypeptide coding sequence, and is recognized by the cell to terminate transcription of the polypeptide coding sequence.

The polyadenylation sequence is a sequence which is operably linked to the 3' terminus of the DNA sequence and which, when transcribed, is recognized by the cell as a signal to add polyadenosine residues to the transcribed mRNA.

The enhancer sequence is a sequence which can increase transcription from a gene when located up to several kilobases from the gene. The enhancer sequencer is usually upstream of the gene.

The attenuator sequence is a sequence which regulates the expression of a gene by determining whether the mRNA molecule containing its transcript will be completed or not.

The intron sequence is a sequence of a gene which is not represented in the protein product of the gene. Intron sequences are transcribed into RNA and must be excised and the RNA molecule religated through a process called intron splicing before it can be translated.

The locus may be noncontiguous or contiguous with the above-noted sequences. Preferably the locus is noncontiguous. The locus may be on the same chromosome or the same extrachromosomal element or on a different chromosome or a different extrachromosomal element as that of the DNA sequence of interest. Furthermore, the locus may be native or foreign to the cell.

In a preferred embodiment, the locus is at least 1,000 bp, more preferably at least 2,000 bp, and even more preferably at least 3,000 bp, even more preferably at least 4,000 bp, even more preferably at least 5,000 bp, and most preferably at least 10,000 bp from the 5' or 3' terminus of the DNA sequence of interest.

In another preferred embodiment, the locus is on a different chromosome than the DNA sequence encoding the polypeptide of interest.

In various methods of the present invention, the nucleic acid constructs are introduced at a locus not within a DNA sequence encoding a protease capable of hydrolyzing the polypeptide under physiological conditions, which means that the nucleic acid construct is not introduced into the protease-coding sequence, the control sequences thereof, any intron sequences within the coding sequence, and any DNA sequences encoding proteins that positively regulate transcription, translation or secretion of the protease.

In another preferred embodiment, the locus encodes a polypeptide different from the polypeptide encoded by the DNA sequence.

In another preferred embodiment, the locus encodes a glucose transporter. Preferably, the locus has at least 60% homology, more preferably at least 70% homology, even more preferably at least 80% homology, even more preferably at least 90% homology, and most preferably at least 95% homology with the nucleic acid sequence of SEQ ID NO:9.

In another preferred embodiment, the locus encodes a mannitol-1-phosphate dehydrogenase. Preferably, the locus has at least 60% homology, more preferably at least 70% homology, even more preferably at least 80% homology, even more preferably at least 90% homology, and most preferably at least 95% homology with the nucleic acid sequence of SEQ ID NO:25.

In another preferred embodiment, the locus encodes a chitin synthase. Preferably, the locus has at least 60% homology, more preferably at least 70% homology, even more preferably at least 80% homology, even more preferably at least 90% homology, and most preferably at least 95% homology with the nucleic acid sequence of SEQ ID NO:56.

In another preferred embodiment, the locus encodes a heat shock protein. Preferably, the locus has at least 60% homology, more preferably at least 70% homology, even more preferably at least 80% homology, even more preferably at least 90% homology, and most preferably at least 95% homology with the nucleic acid sequence of SEQ ID NO:50.

In another preferred embodiment, the locus encodes a manganese superoxide dismutase. Preferably, the locus has at least 60% homology, more preferably at least 70% homology, even more preferably at least 80% homology, even more preferably at least 90% homology, and most preferably at least 95% homology with the nucleic acid sequence of SEQ ID NO:29.

In another preferred embodiment, the locus is a gene required for activation of pacC, preferably a palB gene. Preferably, the locus has at least 60% homology, more preferably at least 70% homology, even more preferably at least 80% homology, even more preferably at least 90% homology, and most preferably at least 95% homology with the nucleic acid sequence of SEQ ID NO:16.

In another preferred embodiment, the locus has at least 60% homology, more preferably at least 70% homology, even more preferably at least 80% homology, even more preferably at least 90% homology, and most preferably at least 95% homology with the nucleic acid sequence of SEQ ID NO:34.

In another preferred embodiment, the locus has at least 60% homology, more preferably at least 70% homology, even more preferably at least 80% homology, even more preferably at least 90% homology, and most preferably at least 95% homology with the nucleic acid sequence of SEQ ID NO:39.

In another preferred embodiment, the locus has at least 60% homology, more preferably at least 70% homology, even more preferably at least 80% homology, even more preferably at least 90% homology, and most preferably at least 95% homology with the nucleic acid sequence of SEQ ID NO:63.

In another preferred embodiment, the locus has at least 60% homology, more preferably at least 70% homology, even more preferably at least 80% homology, even more preferably at least 90% homology, and most preferably at least 95% homology with the nucleic acid sequence of SEQ ID NO:66.

In another preferred embodiment, the locus has at least 60% homology, more preferably at least 70% homology, even more preferably at least 80% homology, even more preferably at least 90% homology, and most preferably at least 95% homology with the nucleic acid sequence of SEQ ID NO:71.

In another preferred embodiment, the locus has at least 60% homology, more preferably at least 70% homology, even more preferably at least 80% homology, even more preferably at least 90% homology, and most preferably at least 95% homology with the nucleic acid sequence of SEQ ID NO:76.

In another preferred embodiment, the locus encodes an aminopeptidase.

In another preferred embodiment, the locus encodes an amylase.

In another preferred embodiment, the locus encodes a carbohydrase.

In another preferred embodiment, the locus encodes a carboxypeptidase.

In another preferred embodiment, the locus encodes a catalase.

In another preferred embodiment, the locus encodes a catalase.

In another preferred embodiment, the locus encodes a cellulase.

In another preferred embodiment, the locus encodes a chitinase.

In another preferred embodiment, the locus encodes a cutinase.

In another preferred embodiment, the locus encodes a deoxyribonuclease.

In another preferred embodiment, the locus encodes a dextranase.

In another preferred embodiment, the locus encodes an esterase.

In another preferred embodiment, the locus encodes an alpha-galactosidase.

In another preferred embodiment, the locus encodes a beta-galactosidase.

In another preferred embodiment, the locus encodes a glucoamylase.

In another preferred embodiment, the locus encodes an alpha-glucosidase.

In another preferred embodiment, the locus encodes a beta-galactosidase.

In another preferred embodiment, the locus encodes a glucoamylase.

In another preferred embodiment, the locus encodes an alpha-glucosidase.

In another preferred embodiment, the locus encodes a beta-glucosidase.

In another preferred embodiment, the locus encodes a haloperoxidase.

In another preferred embodiment, the locus encodes an invertase.

In another preferred embodiment, the locus encodes a laccase.

In another preferred embodiment, the locus encodes a lipase.

In another preferred embodiment, the locus encodes a mannosidase.

In another preferred embodiment, the locus encodes a mutanase.

In another preferred embodiment, the locus encodes an oxidase.

In another preferred embodiment, the locus encodes a pectinolytic enzyme.

In another preferred embodiment, the locus encodes a peroxidase.

In another preferred embodiment, the locus encodes a phytase.

In another preferred embodiment, the locus encodes a polyphenoloxidase.

In another preferred embodiment, the locus encodes a proteolytic enzyme.

In another preferred embodiment, the locus encodes a ribonuclease.

In another preferred embodiment, the locus encodes a transglutaminase.

In another preferred embodiment, the locus encodes a xylanase.

In a more preferred embodiment, the locus is the sequence contained in pDSY109.

In a more preferred embodiment, the locus is the sequence contained in pDSY112.

In a more preferred embodiment, the locus is the sequence contained in pDSY138.

In a more preferred embodiment, the locus is the sequence contained in pDSY141.

In a more preferred embodiment, the locus is the sequence contained in pDSY162.

In a more preferred embodiment, the locus is the sequence contained in pMT1936.

In a more preferred embodiment, the locus is the sequence contained in pSMO1204.

In a more preferred embodiment, the locus is the sequence contained in pSMOH603.

In a more preferred embodiment, the locus is the sequence of SEQ ID NO:9.

In a more preferred embodiment, the locus is the sequence of SEQ ID NO:16.

In a more preferred embodiment, the locus is the sequence of SEQ ID NO:25.

In a more preferred embodiment, the locus is the sequence of SEQ ID NO:29.

In a more preferred embodiment, the locus is the sequence of SEQ ID NO:34.

In a more preferred embodiment, the locus is the sequence of SEQ ID NO:39.

In another more preferred embodiment, the locus is the sequence contained in p4-8.1.

In another more preferred embodiment, the locus is the sequence contained in p7-14.1.

In another more preferred embodiment, the locus is the sequence contained in pHB220.

In another more preferred embodiment, the locus is the sequence contained in pSMO717.

In another more preferred embodiment, the locus is the sequence contained in pSMO321.

In another more preferred embodiment, the locus is the sequence contained in pHowB571.

In another more preferred embodiment, the locus is the sequence contained in pSMO810.

In another more preferred embodiment, the locus is the sequence of SEQ ID NO:50.

In another more preferred embodiment, the locus is the sequence of SEQ ID NO:56.

In another more preferred embodiment, the locus is the sequence of SEQ ID NO:63.

In another more preferred embodiment, the locus is the sequence of SEQ ID NO:66.

In another more preferred embodiment, the locus is the sequence of SEQ ID NO:71.

In another more preferred embodiment, the locus is the sequence of SEQ ID NO:76.

In another preferred embodiment, the locus does not encode a trans factor of the DNA sequence of interest. A "trans factor" is a factor which is encoded by a gene separate from the DNA sequence of interest which activates or represses transcription of the DNA sequence. In a more preferred embodiment, the locus does not encode a repressor of the DNA sequence of interest. In a more preferred embodiment, the locus does not encode an activator of the DNA sequence of interest.

Cells

The methods of the present invention may be used with any cell containing a DNA sequence encoding a polypeptide of interest including prokaryotic cells such as bacteria, or eukaryotic cells such as mammalian, insect, plant, and fungal cells. The DNA sequence may be native or foreign to the cell. The cell may be a unicellular microorganism or a non-unicellular microorganism. Furthermore, the cell may be wild-type or a mutant cell. For example, the mutant cell may be a cell which has undergone classical mutagenesis or genetic manipulation.

Useful prokaryotic cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus, or gram negative bacteria such as E. coli and Pseudomonas sp. In a preferred embodiment, the bacterial cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, or Bacillus subtilis cell.

In a preferred embodiment, the cell is a fungal cell. "Fungi" as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra). Representative groups of Ascomycota include, e.g., Neurospora, Eupenicillium (=Penicillium), Emericella (=Aspergillus), Eurotium (=Aspergillus), and the true yeasts. Examples of Basidiomycota include mushrooms, rusts, and smuts. Representative groups of Chytridiomycota include, e.g., Allomyces, Blastocladiella, Coelomomyces, and aquatic fungi. Representative groups of Oomycota include, e.g., Saprolegniomycetous aquatic fungi (water molds) such as Achlya. Examples of mitosporic fungi include Alternaria, Aspergillus, Candida, and Penicillium. Representative groups of Zygomycota include, e.g., Mucor and Rhizopus.

In a preferred embodiment, the fungal cell is a yeast cell. "Yeast" as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). The ascosporogenous yeasts are divided into the families Spermophthoraceae and Saccharomycetaceae. The latter is comprised of four subfamilies, Schizosaccharomycoideae (e. g., genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e.g., genera Kluyveromyces, Pichia, and Saccharomyces). The basidiosporogenous yeasts include the genera Filobasidiella, Filobasidium, Leucosporidim, Rhodosporidium, and Sporidiobolus. Yeast belonging to the Fungi Imperfecti are divided into two families, Sporobolomycetaceae (e.g., genera Bullera and Sorobolomyces) and Cryptococcaceae (e.g., genus Candida). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner et al., 1980, Soc. App. Bacteriol. Symposium Series No. 9, 1980. The biology of yeast and manipulation of yeast genetics are well known in the art (see, e.g., Biochemistry and Genetics of Yeast, (Bacil, M., Horecker, B. J., and Stopani, A. O. M., editors), 2nd edition, 1987; The Yeasts (Rose, A. H., and Harrison, J. S., editors), 2nd edition, 1987; and The Molecular Biology of the Yeast Saccharomyces, Strathern et al., editors, 1981).

In a more preferred embodiment, the yeast cell is a cell of a species of Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia.

In a most preferred embodiment, the yeast cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis cell. In another most preferred embodiment, the yeast cell is a Kluyveromyces lactis cell. In another most preferred embodiment, the yeast cell is a Yarrowia lipolytica cell.

In another preferred embodiment, the fungal cell is a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative. In a more preferred embodiment, the filamentous fungal cell is a cell of a species of, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia, Tolypocladium, and Trichoderma.

In an even more preferred embodiment, the filamentous fungal cell is an Aspergillus cell. In another even more preferred embodiment, the filamentous fungal cell is an Acremonium cell. In another even more preferred embodiment, the filamentous fungal cell is a Fusarium cell. In another even more preferred embodiment, the filamentous fungal cell is a Humicola cell. In another even more preferred embodiment, the filamentous fungal cell is a Mucor cell. In another even more preferred embodiment, the filamentous fungal cell is a Myceliophthora cell. In another even more preferred embodiment, the filamentous fungal cell is a Neurospora cell. In another even more preferred embodiment, the filamentous fungal cell is a Penicillium cell. In another even more preferred embodiment, the filamentous fungal cell is a Thielavia cell. In another even more preferred embodiment, the filamentous fungal cell is a Tolypocladium cell. In another even more preferred embodiment, the filamentous fungal cell is a Trichoderma cell.

In a most preferred embodiment, the filamentous fungal cell is an Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae cell. In another most preferred embodiment, the filamentous fungal cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, or Fusarium venenatum cell. In a most preferred embodiment, the filamentous fungal cell is a Fusarium venenatum cell (Nirenberg sp. nov.). In another most preferred embodiment, the filamentous fungal cell is a Humicola insolens cell or a Humicola lanuginosa cell. In another most preferred embodiment, the filamentous fungal cell is a Mucor miehei cell. In another most preferred embodiment, the filamentous fungal cell is a Myceliophthora thermophila cell. In another most preferred embodiment, the filamentous fungal cell is a Neurospora crassa cell. In another most preferred embodiment, the filamentous fungal cell is a Penicillium purpurogenum cell. In another most preferred embodiment, the filamentous fungal cell is a Thielavia terrestris cell. In another most preferred embodiment, the filamentous fungal cell is a Trichoderma harzianum, Trichoderma koningii, Trichodenna longibrachiatum, Trichodenna reesei, or Trichoderma viride cell.

Useful mammalian cells include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, or any number of immortalized cells available, e.g., from the American Type Culture Collection.

Introduction of Nucleic Acid Constructs into Cells

The nucleic acid construct(s) may be introduced into a cell by a variety of physical or chemical methods known in the art including, but not limited to, transfection or transduction, electroporation, microinjection, microprojectile bombardment, alkali salts, or protoplast-mediated transformation.

The introduction of the nucleic acid construct into a cell for insertional mutagenesis is referred to as "DNA-tagged mutagenesis". "DNA-tagged mutagenesis" is defined herein as the introduction of a nucleic acid molecule into a cell, which leads to one or more insertions of the nucleic acid molecule into one or more loci of the genome of the cell thereby marking the loci into which the nucleic acid molecule is inserted. The mutant cell produced by DNA-tagged mutagenesis is called a tagged mutant.

Suitable procedures for transformation of Aspergillus cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81: 1470-1474. A suitable method of transforming Fusarium species is described by Malardier et al., 1989, Gene 78: 147-156 or in WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Guide to Yeast Genetics and Molecular Biology, Methods of Enzymology 194: 182-187; Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.

The transformation of a bacterial cell may, for instance, be accomplished by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), by using competent cells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169: 5771-5278).

Mammalian cells may be transformed by direct uptake using the calcium phosphate precipitation method of Graham and Van der Eb, 1978, Virology 52: 546. Other processes, e.g., electroporation, known to the art, may be used.

When the nucleic acid construct is a vector, integration into the cell's genome occurs randomly by homologous and/or non-homologous recombination depending on the cell of choice.

In a preferred embodiment, the nucleic acid construct is introduced into the parent cell by restriction enzyme-mediated integration (REMI). REMI, which is described in Schiestl and Petes, 1991, Proceedings of the National Academy of Sciences USA 88: 7585-7589, is the introduction of plasmid DNA digested with a restriction enzyme along with the restriction enzyme into a cell which subsequently leads to integration of the plasmid DNA into the genome often at a site specified by the restriction enzyme added. The advantage of REMI DNA-tagged mutagenesis is it can generate mutations whose molecular basis can be easily identified.

When the nucleic acid construct is a restriction enzyme cleaved linear DNA fragment, insertion of the construct into the cell's genome through REMI in the presence of the appropriate restriction enzyme is random by virtue of the randomness of the restriction sites present in the genome. The nucleic acid construct may insert into the cell's genome as a single copy or as multiple copies at a single locus or at a different locus or at different loci. It is preferable that the nucleic acid construct insert as a single copy to facilitate the identification and recovery of the tagged locus.

Screening of Mutant Cells

The present invention also relates to mutant cells which produce, express, synthesize or secrete more of a polypeptide or metabolite than the parent cell when both cells are cultivated under the conditions.

The present invention also relates to mutant cells which produce, express, synthesize or secrete more of a polypeptide or metabolite than the parent cell when both cells are cultivated under the conditions.

Following the introduction of a nucleic acid construct into a cell, the next step is to isolate the mutant cell with the modified production of a polypeptide from a population of presumptive mutant cells. The isolation of the mutant cell preferably relies on measurement of the production of the polypeptide or the metabolite by the mutant cell relative to the parent cell when the mutant cell and the parent cell are cultured under the same conditions.

The phrase "modified production of a polypeptide" includes an alteration or change of a step in the production of a polypeptide or a metabolite by the mutant cell relative to the parent cell. Such steps include, but are not limited to, transcription, post-transcriptional modification, translation, post-translational modification, secretion, fermentation, proteolysis, down-stream processing, recovery, and purification.

The mutant cell may be a mutant cell, for example, with improved production of a specific polypeptide or metabolite or a mutant cell which is no longer capable or has a diminished capability of producing a specific polypeptide or metabolite. Furthermore, the mutant cell may be a mutant cell having an increased uptake of an inorganic cofactor.

The mutant cell may also have a more desirable phenotype than the parent cell which modifies the production of a polypeptide or a metabolite. The term "phenotype" is defined herein as an observable or outward characteristic of a cell determined by its genotype and modulated by its environment. Such a mutant cell having a desired phenotype includes, but is not limited to, a morphological mutant cell, a secretion mutant cell, an auxotrophic mutant cell, a conditional mutant, a mutant cell exhibiting an altered growth rate under desired conditions relative to the parent cell, a mutant cell resulting in the relief of overexpression mediated growth inhibition, or a mutant cell able to tolerate low oxygen conditions.

Furthermore, the mutant cell may be characterized as being a mutant cell exhibiting altered production of a transcriptional activator of a promoter or a cryptic intron-splicing-deficient mutant cell.

The isolation of a mutant cell may involve screening methods known in the art specific to the desired phenotype and/or the polypeptide or the metabolite of interest. In general where a desired phenotype is involved, a method specific to the desired phenotype may be used initially to identify the mutant cell, but then may be followed by a method specific to the polypeptide or the metabolite.

The population of presumptive mutants obtained by introducing a nucleic acid construct into the cells of an organism to produce a mutant cell are first purified using standard plating techniques such as those used in classical mutagenesis (see, for example, Lawrence, C. W., 1991, In Christine Guthrie and Gerald R. Fink, editors, Methods in Enzymology, Volume 194, pages 273-281, Academic Press, Inc., San Diego), single spore isolation, or enrichment techniques. The standard plating techniques are preferably conducted in combination with a means of detecting the desired phenotype and/or the polypeptide or the metabolite. Different enrichment techniques may be used for increasing the percentage of mutant cells in comparison to their wild-type or parent equivalents such as (1) direct selection which utilizes growth conditions that greatly favor the growth of the mutant; (2) counterselection, which makes use of conditions that kill the parent cells; (3) physical selection, which involves unique properties of the mutant cells that enable them to be physically separated from their parent cells; and (4) direct measurements of the amount of desired substances. However, whether or not a means for identifying the mutant cell with respect to the desired phenotype and/or the polypeptide or the metabolite of interest can be incorporated into the plating medium, the purified presumptive mutants may require further characterization to confirm the identity of the mutant. Examples of the methods used to further characterize and confirm the identity of the mutant are illustrated below.

A mutant with improved production of a specific polypeptide or a specific metabolite may be identified by using a detection method known in the art that is specific for the polypeptide or the metabolite. Detection methods for polypeptides may include, but are not limited to, use of specific antibodies, enzymatic activity by measuring formation of an enzyme product or disappearance of an enzyme substrate, clearing zones on agar plates containing an enzyme substrate, and biological activity assays. Detection methods for metabolites may include, but are not limited to, thin layer chromatography, high performance liquid chromatography, gas chromatography, mass spectroscopy, biological activity assays, bioassays, and fluorescent activating cell sorting.

In a preferred embodiment, the specifically desired mutant cell is a mutant cell with improved production of a specific polypeptide.

In another preferred embodiment, the specifically desired mutant is a mutant with improved production of a specific metabolite; more preferably an alkaloid, an amino acid, an antibiotic, a cofactor, a drug, a fatty acid, a fungicide, a herbicide, an insecticide, an organic acid, a pigment, a plastic precursor, a polyester precursor, a prosthetic group, a rodenticide, a sweetner, or a vitamin; and most preferably citric acid or lactic acid.

A prosthetic group or an organic cofactor which is a constituent of a polypeptide and/or required for biological activity may be overproduced by isolating a mutant according to the methods of the present invention. Such a mutant would be particularly important where biosynthesis of the prosthetic group or the cofactor is a rate-limiting event in the production of a polypeptide in a biologically active form, e.g., a hemoprotein containing heme including, but not limited to, a cytochrome, specifically cytochrome P450, cytochrome b, cytochrome c₁, or cytochrome c; a globin, specifically, hemoglobin or myoglobin; an oxidoreductase, specifically a catalase, an oxidase, an oxygenase, a haloperoxidase, or a peroxidase; or any other polypeptide containing a heme as a prosthetic group.

In a more preferred embodiment, the specifically desired mutant cell is a mutant cell overproducing an adenosine phosphate, S-adenosyl-L-methionine, biocytin, biotin, coenzyme A, coenzyme Q (ubiquinone), 5'-deoxyadenosylcobalamine, a ferredoxin, a flavin coenzyme, heme, lipoic acid, a nucleoside diphosphate, a nicotinamide adenine dinucleotide, a nicotinamide adenine dinucleotide phosphate, phosphoadenosine, phosphosulfate, pyridoxal phosphate, tetrahydrofolic acid, thiamine pyrophosphate, or a thioredoxin.

In another preferred embodiment, the specifically desired mutant cell is a mutant cell characterized with an increased uptake of an inorganic cofactor. The uptake by a cell of an inorganic cofactor which is a constituent of a polypeptide and/or required for biological activity may be increased by isolating a mutant according to the methods of the present invention. Such a mutant would be particularly important where uptake of the inorganic cofactor is a rate-limiting event in the production of a polypeptide in a biologically active form. In a more preferred embodiment, the specifically desired mutant cell is a mutant cell characterized with an increased uptake of Co²⁺, Cu²⁺, Fe²⁺, Fe³⁺, K⁺, Mg²⁺, Mn²⁺, Ni²⁺, Se, or Zn²⁺.

In a preferred embodiment, the polypeptide or the metabolite is produced by the mutant cell in an amount which is at least 20% greater, preferably at least 50% , more preferably at least 75% , more preferably at least 100% , more preferably at least 100%-1000%, even more preferably at least 200%-1000% , and most preferably at least 500%-1000% or more greater than the cell.

In another preferred embodiment, the specifically desired mutant cell is a mutant cell which is no longer capable or has a diminished capability of producing a specific polypeptide. A mutant cell which is no longer capable or has a diminished capability of producing a specific polypeptide may be identified using the same methods described above for polypeptides, but where no or diminished production is measured relative to the parent cell.

In a more preferred embodiment, the specifically desired mutant cell is a mutant cell which is no longer capable or has a diminished capability of producing a polypeptide.

In another preferred embodiment, the polypeptide is produced by the mutant cell in an amount which is at least 20% , more preferably at least 50% , even more preferably at least 75% , and most preferably 100% lower than the cell.

In another preferred embodiment, the specifically desired mutant cell is a mutant cell which is no longer capable or has a diminished capability of producing a specific metabolite. A mutant cell which is no longer capable or has a diminished capability of producing a specific metabolite may be identified using the same or similar methods described above for metabolites, but where no or diminished production is measured relative to the parent cell.

In a more preferred embodiment, the specifically desired mutant cell is a mutant cell which is no longer capable or has a diminished capability of producing a deoxysugar, a surfactant, a mycotoxin, an organic acid, a sugar alcohol, a toxic metabolite, or a toxin; and most preferably an aflatoxin, beta-exotoxin, cyclopiazonic acid, an enniatin, a fusarin, kanosamine, mannitol, oxalic acid, surfactin, a tricothecene, a zearalenol, or a zearalenone.

In another preferred embodiment, the metabolite is produced by the mutant cell in an amount which is at least 20% lower than the cell, more preferably 50% , even more preferably 75% , and most preferably 100% lower than the cell.

In another preferred embodiment, the mutant cell is a morphological mutant cell. A "morphological mutant cell" is defined herein as a mutant cell which has a desired morphology. A morphological mutant cell may be identified, for example, by using standard plating techniques employing a growth medium which elicits the desired morphology relative to the parent cell, by microscopic examination, or by sorting vegetatively growing cells by fluorescence activated cell sorting. Such morphological mutants include, but are not limited to, a mutant characterized as having superior rheological properties, e.g., a highly-branched fungal mutant, a restricted colonial fungal mutant, or a highly-branched restricted colonial fungal mutant which possesses rapid growth and low viscosity growth characteristics; a mutant which possesses a filamentous form during fermentation in contrast to a pellet form; a mutant which is less "sticky" preventing the colonization of fermentor surfaces; a mutant with a predictable viscosity during the course of a fermentation; a color mutant which aids in monitoring and maintaining the purity of a culture and high production of a polypeptide by the culture; a wettable cell which lacks, for example, a cell wall or structural hydrophobic protein, e.g., hydrophobin; an osmotic stress-insensitive mutant which improves growth of a cell; a desiccation-insensitive mutant which improves growth of a cell; a non-spore-forming mutant which enhances the production of a polypeptide; and a non-slime-producing mutant with low viscosity growth.

Preferably, the morphological mutant cell is a color mutant, a wettable mutant cell, a mutant characterized as having superior rheological properties, an osmotic stress-insensitive mutant, a desiccation-insensitive mutant, a non-spore-forming mutant, or a non-slime-producing mutant, and most preferably a highly-branched fungal mutant, a restricted colonial fungal mutant, or a highly-branched restricted colonial fungal mutant.

In another preferred embodiment, the mutant cell is a secretion mutant cell. A "secretion mutant cell" is defined herein as a mutant cell which produces higher yields of one or more secreted proteins. A secretion mutant cell may be identified by using a detection method known in the art that is specific for the polypeptide and comparing the yield to one or more known secreted polypeptides at the same time. Detection methods for polypeptides may include, but are not limited to, use of specific antibodies, enzymatic activity by measuring formation of an enzyme product or disappearance of an enzyme substrate, clearing zones on agar plates containing an enzyme substrate, biological activity assays, and fluorescent activating cell sorting.

In another preferred embodiment, the specifically desired mutant cell is an auxotrophic mutant cell. An "auxotrophic mutant cell" is defined herein as a mutant cell which has lost its ability to synthesize one or more essential metabolites or to metabolize one or more metabolites which modifies the production of a polypeptide by the mutant cell. An auxotrophic mutant cell may be identified using standard plating techniques by growing the presumptive mutant both in the absence and presence of an essential metabolite. The auxotrophic mutant will not grow in the absence of the essential metabolite. The auxotrophic mutant can be advantageously used to selectively screen for a mutant producing a specific polypeptide of interest.

In a more preferred embodiment, the specifically desired mutant cell is an auxotrophic mutant cell unable to metabolize or synthesize one or more of an amino acid, a fatty acid, an organic acid, a pyrimidine, a purine, or a sugar; and more preferably 5-aminolevulinic acid, biotin, glucose, lactose, or maltose.

In another preferred embodiment, the specifically desired mutant cell is a conditional mutant cell. A "conditional mutant cell" is defined herein as a mutant cell which contains one or more mutations whose phenotypes are only observed under certain conditions and modifies the production of a polypeptide or a metabolite by the mutant cell. Conditional mutations can occur in virtually all genes, including those that control the steps in macromolecular synthesis, modification, and assembly into supermolecular structures. A conditional mutant cell may be identified using standard plating techniques by growing the presumptive mutant both under permissive and restrictive conditions. For example, a mutant strain which does not produce undesirable proteolytic activity under nitrogen limited conditions would be desirable compared to the parent strain which produces proteolytic activity under nitrogen limited conditions. An additional example is an alkaline pH sensitive mutant that does not grow at alkaline pH, but may have increased or decreased production of a desired polypeptide. A further example is a mutant which is unable to grow under specific growth conditions.

In a more preferred embodiment, the conditional mutant cell is a temperature sensitive, acid pH sensitive, alkaline pH sensitive, antibiotic-resistant, antibiotic-sensitive, toxin-resistant, toxin-sensitive, virus-resistant, or paraquat-sensitive cell; and most preferably an alkaline pH sensitive mutant cell.

In another preferred embodiment, the specifically desired mutant cell is a mutant cell exhibiting an altered growth rate relative to the parent cell. A "mutant cell exhibiting an altered growth rate" is defined herein as a mutant cell which has a doubling time that is different than that of the parent cell. Such a mutant cell may be identified by comparing the growth of the mutant cell and the parent cell under controlled fermentation conditions. Such a mutant cell may have improved fermentation characteristics like a shorter fermentation time to increase productivity, or a longer fermentation time to provide control of the oxygen demand of a culture.

In another preferred embodiment, the specifically desired mutant cell is a mutant cell resulting in the relief of overexpression mediated growth inhibition. A "mutant cell resulting in the relief of overexpression mediated growth inhibition" is defined herein as a mutant cell whose growth is not inhibited by the overproduction of a desired polypeptide or metabolite when grown under conditions that induce high level production of the polypeptide or the metabolite. Such a mutant may be identified by standard plating techniques on plates with an inducing carbon source, e.g., maltose. Mutants would be able to grow well on the inducing carbon source while the parent cells would grow poorly. Such a mutant would be useful since it is known in some cells that overexpression of a polypeptide is toxic to the cells.

In another preferred embodiment, the specifically desired mutant cell is a mutant cell able to tolerate low oxygen conditions. A "mutant cell able to tolerate low oxygen conditions" is defined herein as a mutant cell which is able to grow and produce a desired polypeptide or metabolite under growth conditions where the dissolved oxygen concentration is low. Such a mutant cell is particularly advantageous for fermentations where the productivity of high cell densities decreases due to oxygen transfer. A low oxygen tolerant mutant is preferably detected by growing the mutant cell relative to the parent cell on a solid or in a liquid medium in the presence of low levels of oxygen.

In a more preferred embodiment, the specifically desired mutant cell is a mutant cell able to tolerate low oxygen conditions in the range of about 0 to about 50% saturation, preferably about 0 to about 40% saturation, even more preferably about 0% to about 30% saturation, more preferably about 0% to about 20% saturation, most preferably about 0% to about 10% saturation, and even most preferably about 0% to about 5% saturation.

In another preferred embodiment, the specifically desired mutant cell is a signal transduction pathway mutant cell. A "signal transduction pathway mutant cell" is defined herein as a mutant cell with a mutation in one or more of the genes of the pathway which modifies the production of a polypeptide encoded by a DNA sequence of interest. The term "signal transduction pathway" is defined herein as a cascade of genes encoding polypeptides that are all required for the activation or deactivation of another single polypeptide. The pathway senses a signal and through the cascade of genes, the signal is transduced and leads to the activation or deactivation of one or more polypeptides. Such a mutant is preferably detected using a method which is specific to the desired phenotype which modifies the production of a polypeptide of interest.

In a more preferred embodiment, the signal transduction pathway mutant cell is a glucose transport signal transduction pathway mutant or a pH signal transduction pathway mutant, even more preferably a mutant in which gene required for activation of pacC has been disrupted, and most preferably a gluT gene mutant or a palB gene mutant.

In another preferred embodiment, the specifically desired mutant cell is a mutant cell exhibiting altered production of a transcriptional activator of a promoter. A "mutant cell exhibiting altered production of a transcriptional activator of a promoter" is defined herein as a mutant cell with a mutation in a gene encoding a transcriptional activator which `turns-up` or `turns-down` a promoter of a DNA sequence encoding a polypeptide of interest.

Examples of such promoters in a bacterial cell are promoters of the genes of the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus licheniformis penicillinase gene (penP), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus subtilis levansucrase gene (sacB), the Bacillus subtilis xylA and xylB genes, the E. coli lac operon, the Streptomyces coelicolor agarase gene (dagA), and the prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences USA 80: 21-25). Further promoters are described in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242: 74-94; and in J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York.

Examples of such promoters in a filamentous fungal cell are promoters of the genes encoding Aspergillus nidulans acetamidase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger neutral alpha-amylase, Aspergillus awamori or Aspergillus niger glucoamylase (glaA), Aspergillus oryzae alkaline protease, Aspergillus oryzae TAKA amylase, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (as described in U.S. Pat. No. 4,288,627, which is incorporated herein by reference), Rhizomucor miehei aspartic proteinase, Rhizomucor miehei lipase, and mutant, truncated, and hybrid promoters thereof. Particularly preferred promoters in filamentous fungal cells are the TAKA amylase, NA2-tpi (a hybrid of the promoters from the genes encoding Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and glaA promoters.

Examples of such promoters in a yeast cell are promoters of the genes encoding Saccharomyces cerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiae galactokinase gene (GAL1), the Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP), and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Other yeast promoters are described by Romanos et al., 1992, Yeast 8: 423-488.

Examples of such promoters in a mammalian cell are viral promoters such as those of Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus, and bovine papilloma virus (BPV).

In a more preferred embodiment, the mutant cell exhibits altered production of a transcriptional activator of the TAKA, TAKA/NA2, Fusarium oxysporum trypsin-like protease, or a glucoamylase promoter.

In another preferred embodiment, the specifically desired mutant cell is a cryptic intron-splicing-deficient mutant cell. A "cryptic intron-splicing-deficient mutant cell" is defined herein as a mutant cell which no longer recognizes and erroneously splices a cryptic intron as an authentic intron during mRNA synthesis. A cryptic intron-splicing-deficient mutant cell would be particularly useful to prevent the excision or splicing of an erroneous cryptic nuclear pre-mRNA intron from a primary transcript so a biologically active substance is produced. In this situation, the cryptic intron is actually part of the coding sequence and, therefore, is not an authentic intron but incorrectly recognized as such and erroneously spliced during mRNA synthesis and processed by the parent cell. The introduction of a DNA sequence encoding a heterologous polypeptide into a parent cell such as a fungal host cell, particularly a filamentous fungal host cell, may result in this type of erroneous or aberrant splicing of the coding sequence. A cryptic intron-splicing-deficient mutant cell may be identified by screening for increased production of a polypeptide encoded by a DNA sequence that is known to have a cryptic intron which leads to little or no production of the polypeptide.

In another preferred embodiment, the specifically desired mutant may be a mutant which contains two or more of the mutations described above.

Identification of Mutant Cells of the Present Invention

The present inventors have discovered that when certain loci in a parent cell are disrupted, the resulting mutant cell has a modified production of a polypeptide. As described above, the nucleic acid construct itself can have an effect on the production of a polypeptide. For example, the nucleic acid construct may comprise one or more copies of the nucleic acid sequence encoding the polypeptide. In addition, the nucleic acid construct may comprise a promoter, transcriptional activators and repressors, etc.

When the nucleic acid construct itself can have an effect on the amount of polypeptide produced, expressed, synthesized or secreted, in order to determine whether a mutant cell of the present invention has been produced, one would have to rescue the locus as described below and introduce another nucleic acid construct which does not have an effect, e.g., a selectable marker, at the same locus. If the mutant cell produced by introducing the other nucleic acid construct at the same locus also has an effect on the amount of polypeptide produced, expressed, synthesized or secreted, then the original mutant cell is a mutant cell of the present invention.

Rescue of a Locus with the Inserted Nucleic Acid Construct and Use of a Targeting Construct

The present invention further relates to methods for rescuing a locus with the inserted nucleic acid construct comprising isolating from the identified mutant cell (i) the nucleic acid construct and (ii) the 3' and 5' flanking regions of the locus of the genome where the nucleic acid construct has been integrated; and identifying the 3' and 5' flanking regions of the locus.

The nucleic acid construct and flanking regions can be isolated or rescued by methods well known in the art such as cleaving with restriction enzymes and subsequent ligation and transformation of E. coli, inverse PCR, random primed gene walking PCR, or probing a library of the tagged mutant. The isolated nucleic acid construct with either or both the 3' and 5' flanking regions is defined herein as a "targeting construct".

The targeting construct includes between 100-9,000 bp, preferably 200-9,000 bp, more preferably 500-7,000 bp, even more preferably 1,000-7,000 bp, and most preferably 1,000-3,000 bp upstream and/or downstream of the integration site of the nucleic acid construct.

The targeting construct of the invention may be introduced into a different cell to modify the production of a polypeptide similar or identical to or completely different from the polypeptide modified in the original cell. The other cell may be of the same or a different species or of a different genera as the original cell. If the original cell was a fungal cell, the other cell is preferably a fungal cell. If the original cell was a bacterial cell, the other cell is preferably a bacterial cell. If the original cell was a mammalian cell, the other cell is preferably a mammalian cell.

When the cell is a different cell, integration of the targeting construct preferably occurs at a target locus which is homologous to the locus sequence of the original cell from which the targeting construct was obtained, i.e., identical or sufficiently similar such that the targeting sequence and cellular DNA can undergo homologous recombination to produce the desired mutation. The sequence of the targeting construct is preferably, therefore, homologous to a preselected site of the cellular chromosomal DNA with which homologous recombination is to occur. However, it will be understood by one of ordinary skill in the art that the likelihood of a targeting construct reinserting at a target locus will depend on the cell since homologous recombination frequencies range from almost 100% in the yeast Saccharomyces cerevisiae to as low as 1% in Aspergillus. The targeting construct may integrate by non-homologous recombination at a non-target locus which is not within the DNA sequence encoding the polypeptide of interest, but results in the modification of the production of the polypeptide.

Preferably, the target locus includes DNA sequences that have greater than 40% homology, preferably greater than 60% homology, more preferably greater than 70% homology, even more preferably greater than 80% homology, and most preferably greater than 90% homology with the flanking sequences of the targeting construct.

The targeting construct may contain either or both of the 3' and 5' regions depending on whether a single cross-over or a replacement is desired. Furthermore, the targeting construct may be modified to correct any aberrant events, such as rearrangements, repeats, deletions, or insertions, which occurred during the introduction and integration of the original nucleic acid construct into the cell's genome at the locus from which it was originally rescued.

The targeting construct described above may be used as is, i.e., a restriction enzyme cleaved linear nucleotide sequence, or may be circularized or inserted into a suitable vector. For example, a circular plasmid or DNA fragment preferably employs a single targeting sequence. A linear plasmid or DNA fragment preferably employs two targeting sequences. The targeting construct upon introduction into a cell, in which the cell comprises a DNA sequence encoding a polypeptide of interest, integrates into the genome of the cell at a target locus or at a nontarget locus, but preferably at a target locus, not within the DNA sequence encoding the polypeptide of interest. The target locus may be on the same chromosome or the same extrachromosomal element or on a different chromosome or a different extrachromosomal element as that of the DNA sequence of interest. The integration modifies the production of the polypeptide or a metabolite by the mutant cell relative to the parent cell when the mutant cell and the parent cell are cultured under the same conditions. In a preferred embodiment, the targeting construct contains a selectable marker.

Optionally, the targeting construct can be introduced into a cell as two or more separate fragments. In the event two fragments are used, the fragments share DNA sequence homology (overlap) at the 3' end of one fragment and the 5' end of the other, while one carries a first targeting sequence and the other carries a second targeting sequence. Upon introduction into a cell, the two fragments can undergo homologous recombination to form a single fragment with the first and second targeting sequences flanking the region of overlap between the two original fragments. The product fragment is then in a form suitable for homologous recombination with the cellular target sequences. More than two fragments can be used, designed such that they will undergo homologous recombination with each other to ultimately form a product suitable for homologous recombination with the cellular target sequences.

Upon introduction of the targeting construct into a cell, the targeting construct may be further amplified by the inclusion of an amplifiable selectable marker gene which has the property that cells containing amplified copies of the selectable marker gene can be selected for by culturing the cells in the presence of the appropriate selectable agent.

In a specific embodiment, the targeting construct is SphI linearized pDSY109, HpaI linearized pDSY112, AsnI/PvuI linearized pMT1936, NdeI linearized pDSY138, AsnI/PvuI linearized pDSY162, BglII linearized p4-8.1, BglII linearized p4-8.1, NarI linearized p7-14.1, BglII linearized pSMO717, BglII linearized pSMO321, NdeI linearized pHowB571, or NdeI linearized pSMO810.

In a most preferred embodiment, the nucleic acid construct is pDSY109.

In a most preferred embodiment, the nucleic acid construct is pDSY112.

In a most preferred embodiment, the nucleic acid construct is pMT1936.

In a most preferred embodiment, the nucleic acid construct is pDSY138.

In a most preferred embodiment, the nucleic acid construct is pDSY162.

In a most preferred embodiment, the nucleic acid construct is pDSY163.

In a most preferred embodiment, the nucleic acid construct is pDSY141.

In a most preferred embodiment, the nucleic acid construct is pSMO1204.

In a most preferred embodiment, the nucleic acid construct is pSMOH603.

In a most preferred embodiment, the nucleic acid construct is p4-8.1.

In a most preferred embodiment, the nucleic acid construct is p7-14.1.

In a most preferred embodiment, the nucleic acid construct is pHB220.

In a most preferred embodiment, the nucleic acid construct is pSMO717.

In a most preferred embodiment, the nucleic acid construct is pSMO321.

In a most preferred embodiment, the nucleic acid construct is pHowB571.

In a most preferred embodiment, the nucleic acid construct is pSMO810.

In a preferred embodiment, one or more targeting constructs are introduced into target loci. In another preferred embodiment, each targeting construct modifies the production of a different polypeptide or a different metabolite or a combination thereof, or results in different phenotypes which modify the production of different polypeptides or different metabolites or a combination thereof. In another preferred embodiment, two or more targeting constructs together when introduced into target loci act additively or synergistically to modify the production of a polypeptide or a metabolite.

Methods of Producing a Desired Polypeptide or Metabolite from Mutant Cells

The present invention further relates to the mutant cells with a desired phenotype as host cells. Mutant cells selected for increased production of a desired polypeptide or metabolite are cultivated in a nutrient medium suitable for production of the polypeptide or metabolite using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide or metabolite to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art (see, e.g., references for bacteria and yeast; Bennett, J. W. and LaSure, L., editors, More Gene Manipulations in Fungi, Academic Press, Calif., 1991). Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide or metabolite is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide or metabolite is not secreted, it is recovered from cell lysates.

The polypeptides and metabolites may be detected using methods known in the art that are specific for the polypeptides and metabolites such as those methods described earlier or the methods described in the Examples.

The resulting polypeptide or metabolite may be recovered by methods known in the art. For example, the polypeptide or metabolite may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

The polypeptides and metabolites of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use various constructs and perform the various methods of the present invention and are not intended to limit the scope of what the inventors regard as their invention. Unless indicated otherwise, parts are parts by weight, temperature is in degrees centigrade, and pressure is at or near atmospheric pressure. Efforts have been made to ensure accuracy with respect to numbers used (e.g., length of DNA sequences, molecular weights, amounts, particular components, etc.), but some deviations should be accounted for.

Example 1

Strains and Materials

The starting strains were pyrG-minus Aspergillus oryzae HowB425, pyrG-minus Aspergillus oryzae HowB101, Aspergillus oryzae JaL250, Aspergillus niger strain JRoy3 (pyrGΔ), E. coli DH5α (GIBCO-BRL, Gaithersburg, Md.), and E. coli HB101 (GIBCO-BRL, Gaithersburg, Md.).

PDA plates contained 39 g/l Potato Dextrose Agar (Difco) and were supplemented with 10 mM uridine for pyrG auxotrophs unless otherwise indicated.

MY25 medium at pH 6.5 was composed per liter of 25 g of maltose, 2.0 g of MgSO₄ --7H₂ O, 10 g of KH₂ PO₄, 2.0 g of citric acid, 10 g of yeast extract, 2.0 g of K₂ SO₄, 2.0 g of urea, and 0.5 ml of trace metals solution. MY25 shake-flask medium was diluted 1:100 or 1:1000 with glass distilled water for use in microtiter growth experiments (MY25/100 or MY25/1000). Cultures were grown at 34° C.

2X MY Salts pH 6.5 solution was composed per liter of 4 g of MgSO₄ --7H₂ O, 4 g of K₂ SO₄, 20 g of KH₂ PO₄, 4 g of citric acid, 1 ml of trace metals, and 2 ml of CaCl₂ --2H₂ O (100 g/l stock solution.

Minimal medium transformation plates were composed per liter of 6 g of NaNO₃, 0.52 g of KCl, 1.52 g of KH₂ PO₄, 1 ml of trace metals solution, 1 g of glucose, 500 mg of MgSO₄ --7H₂ O, 342.3 g of sucrose and 20 g of Noble agar per liter (pH 6.5). Minimal medium transfer plates (pH 6.5) were composed per liter of 6 g of NaNO₃, 0.52 g of KCl, 1.52 g of KH₂ PO₄, 1 ml of trace elements, 1 g of glucose, 500 mg of MgSO₄ --7H₂ O, and 20 g Noble agar.

The trace metals solution (1000×) was composed per liter of 22 g of ZnSO₄ --7H₂ O, 11 g of H₃ BO₃, 5 g of Mncl₂ --4H₂ O, 5 g of FeSO₄ --7H₂ O, 1.6 g of CoCl₂ --5H₂ O, 1.6 g of (NH₄)₆ Mo₇ O₂₄, and 50 g of Na₄ EDTA.

COVE plates were composed per liter of 343.3 g of sucrose, 20 ml of COVE salts solution, 10 ml of 1 M acetamide, 10 ml of 3 M CsCl, and 25 g of Nobel agar. The COVE salts (50×) solution was comprised of 26 g of KCl, 26 g of MgSO₄ --7H₂ O, 76 g of KH₂ PO₄, and 50 ml of COVE trace metals solution. COVE trace metals solution was composed of (per liter): 0.04 g of NaB₄ O₇ --10H₂ O, 0.040 g of CuSO₄ --5H₂ O, 0.70 g of FeSO₄ --H₂ O, 0.08 g of Na₂ MoO₂ --2H₂ O, and 10 g of ZnSO₄.

YEG medium was composed per liter of 5 g yeast extract and 20 g dextrose.

CM-1 agar plates at pH 6.5 were composed per liter of 0.25 g of NaCl, 0.5 g of MgSO₄ --7H₂ O, 1.9 g of K₂ HPO₄, 3.6 g of KH₂ PO₄, 0.1 ml of trace metals solution, 30 g of Bacto agar (Difco), pH 6.5. 11 ml of 10% urea, and 67 ml of 30% maltose.

CD medium was composed per liter of 1 g of MgSO₄ --7H₂ O, 1 g of K₂ SO₄, 15 g of KH₂ PO₄, 0.25 ml of trace metals solution, 0.7 g of yeast extract (Difco), 20 g of beta-cyclodextrin (Sigma C-4767). 3 ml of 50% urea, and 2 ml of 15% CaCl₂ --2H₂ O.

G1-gly medium was composed per liter of 18 g of yeast extract (Difco), 80 g of 75% glycerol, and 0.5 g of CaCl₂ --2H₂ O.

OL-1 medium (pH 7.0) was composed per liter of 15 g of KH₂ PO₄, 1 g of MgSO₄ --7H₂ O, 1 g of K₂ SO₄, 0.25 ml of trace metals solution, 0.3 g of CaCl₂ --2H₂ O (autoclaved separately), 2 g of Difco yeast extract (Difco), 0.5 g of urea (autoclaved separately), and 10 g of glucose.

OL-6 medium (pH 7.0) was composed per liter of 15 g of KH₂ PO₄, 1 g of MgSO₄ --7H₂ O, 1 g of K₂ SO₄, 0.25 ml of trace metals solution, 0.3 g of CaCl₂ --2H₂ O (autoclaved separately), 2 g of Difco yeast extract (Difco), 3 g of urea (autoclaved separately), and 60 g of glucose.

YPM medium was composed of 10 g of Bactopeptone and 5 g of yeast extract dissolved in 500 ml of water and autoclaved, to which 50 ml of a sterilized 20% maltose solution was added.

MTBCDUY was composed per liter of 0.3 g of MgSO₄ --7H₂ O, 0.3 g of K₂ SO₄, 5 g of KH₂ PO₄, 0.013 g of urea, 0.01 g of yeast extract, 0.1 g of maltose, 4.88 g of uridine, and 0.25 ml of trace metal solution 1 adjusted to pH 6.5.

4xMTBCDUY was composed per liter of 0.3 g of MgSO₄ --7H₂ O, 0.3 g of K₂ SO₄, 5 g of KH₂ PO₄, 0.052 g of urea, 0.04 g of yeast extract, 0.4 g of maltose, 4.88 g of uridine, and 0.25 ml of trace metal solution 1.

MDU1B was composed per liter of 45 g of Maltodextrin MD01, 1.0 g of MgSO₄ --7H₂ O, 1.0 g of NaCl, 2.0 g of K₂ SO₄, 12.0 g of KH2PO₄, 7.0 g of yeast extract, 0.5 ml of trace metal solution, and 0.1 ml of pluronic acid. The trace metal solution consisted of 13.9 g of FeSO₄ --7H₂ O, 8.45 g of MnSO₄ --H₂ O, 6.8 g of ZnCl₂, 2.5 g of CuSO₄ --5H₂ O, 2.5 g of NiCl₂ --6H₂ O, and 3 g of citric acid. The pH of the shake flask medium was adjusted to 5.0 before being autoclaved.

1/5 MDU2BP was composed per liter of 9 g of maltose, 0.2 g of MgSO₄ --7H₂ O, 0.4 g of K₂ SO₄, 0.2 g of NaCl, 2.4 g of KH₂ PO₄, 1.0 g of urea, 1.4 g of yeast extract, and 0.1 ml of trace metal solution 1.

Trace metal solution 1 was composed per liter of 13.8 g of FeSO₄ --7H₂ O, 8.5 g of MnSO₄ H₂ O, 14.3 g of ZnSO₄ --7H₂ O, 2.5 g of CuSO₄ --5H₂ O, 0.5 g of NiCl₂ --6H₂ O, and 3.0 g of citric acid.

YPG plates was composed per liter of 4.0 g yeast extract, 1.0 g of K₂ HPO₄, 0.5 g of MgSO₄ --7H₂ O, 15.0 g of dextrose, and 20.0 g of agar.

Example 2

Construction of Aspergillus oryzae HowB430

Aspergillus oryzae HowB430 was constructed to contain a lipase gene from Humicola lanuginosa (LIPOLASE™ gene, Novo Nordisk A/S, Bagsv.ae butted.rd, Denmark).

pBANe8 was constructed as described below to contain the TAKA/NA2-tpi leader hybrid promoter, the lipase gene from Humicola lanuginosa, the AMG terminator, and the full-length Aspergillus nidulans amdS gene as a selectable marker.

PCR was employed to insert NsiI sites flanking the full-length amdS gene of pToC90 (Christensen et al., 1988, Biotechnology 6: 1419-1422) using primers 1 and 2 below and to insert an EcoRI site at the 5' end and a SwaI site at the 3' end of the NA2-tpi leader hybrid promoter of pJaL292 (FIG. 1) using primers 3 and 4 below. The primers were synthesized with an Applied Biosystems Model 394 DNA/RNA Synthesizer (Applied Biosystems, Inc., Foster City, Calif.) according to the manufacturer's instructions.

Primer 1: 5'-ATGCATCTGGAAACGCAACCCTGA-3' (SEQ ID NO:1)

Primer 2: 5'-ATGCATTCTACGCCAGGACCGAGC-3' (SEQ ID NO:2)

Primer 3: 5'-TGGTGTACAGGGGCATAAAAT-3' (SEQ ID NO:3)

Primer 4: 5'-ATTTAAATCCAGTTGTGTATATAGAGGATTGTGG-3' (SEQ ID NO:4)

Amplification reactions (100 μl) were prepared using approximately 0.2 μg of either pToC90 or pJaL292 as the template. Each reaction contained the following components: 0.2 μg of plasmid DNA, 48.4 pmol of the forward primer, 48.4 pmol of the reverse primer, 1 mM each of dATP, dCTP, dGTP, and dTTP, 1×Taq polymerase buffer, and 2.5 U of Taq polymerase (Perkin-Elmer Corp., Branchburg, N.J.). The reactions were incubated in an Ericomp Thermal Cycler programmed as follows: One cycle at 95° C. for 5 minutes followed by 30 cycles each at 95° C. for 1 minute, 55° C. for 1 minute and 72° C. for 2 minutes.

The PCR products were electrophoresed on a 1% agarose gel to confirm the presence of a 2.7 kb amdS fragment and a 0.6 kb NA2-tpi fragment.

The PCR products were subsequently subcloned into pCRII using a TA Cloning Kit (Invitrogen, San Diego, Calif.) according to the manufacturer's instructions. The transformants were then screened by extracting plasmid DNA from the transformants using a QIAwell-8 Plasmid Kit (Qiagen, Inc., Chatsworth, Calif.) according to the manufacturer's instructions, and restriction digesting the plasmid DNA with either NsiI or EcoRI/SwaI followed by agarose electrophoresis to confirm the presence of the correct size fragments, 2.7 kb and 0.6 kb, respectively, for the NsiI amdS fragment and SwaI/EcoRI NA2-tpi fragment. In order to confirm the PCR products, the products were sequenced with with an Applied Biosystems Model 373A Automated DNA Sequencer (Applied Biosystems, Inc., Foster City, Calif.) on both strands using the primer walking technique with dye-terminator chemistry (Giesecke et al., 1992, Journal of Virol. Methods 38: 47-60) using the M13 reverse (-48) and M13 forward (-20) primers (New England Biolabs, Beverly, Mass.) and primers unique to the DNA being sequenced. The plasmids from the correct transformants were then digested with the restriction enzymes for which the plasmids were designed, separated on a 1% agarose gel, and purified using a FMC SpinBind Kit (FMC, Rockland, Me.) according to the manufacturer's instructions.

pKS6 (FIG. 2), which contains the TAKA promoter, a polylinker, the AMG terminator, and the Aspergillus nidulans pyrG gene, was digested with EcoRI and SwaI to remove a portion of the TAKA promoter. This region was replaced with the NA2-tpi PCR product to produce pBANe13 (FIG. 3).

pBANe13 was digested with NsiI to remove the Aspergillus nidulans pyrG gene. This region was then replaced with the full length amdS gene PCR product described above to produce pBANe6 (FIG. 4).

PCR was used to insert SwaI and PacI flanking sites on the full-length Humicola lanuginosa lipase gene of pMHan37 (FIG. 5) using primers 5 and 6 below. Primers 5 and 6 were synthesized as described above.

Primer 5: 5'-ATTTAAATGATGAGGAGCTCCCTTGTGCTG-3' (SEQ ID NO:5)

Primer 6: 5'-TTAATTAACTAGAGTCGACCCAGCCGCGC-3' (SEQ ID NO:6)

The amplification reaction (100 μl) contained the following components: 0.2 μg of pMHan37, 48.4 pmol of primer 5, 48.4 pmol of primer 6, 1 mM each of dATP, dCTP, dGTP, and dTTP, 1×Taq polymerase buffer, and 2.5 U of Taq polymerase. The reaction was incubated in an Ericomp Thermal Cycler programmed as follows: One cycle at 95° C. for 5 minutes followed by 30 cycles each at 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 2 minutes. Two μl of the reaction was electrophoresed on an agarose gel to confirm the amplification of the lipase gene product of approximately 900 bp.

The PCR amplified lipase gene product was then subcloned into pCRII using a TA Cloning Kit. The transformants were screened by extracting plasmid DNA from the transformants using a QIAwell-8 Plasmid Kit, restriction digesting the plasmid DNA with SwaI/PacI, and sequencing the DNA according to the method described above to confirm the PCR product.

The lipase gene was excised from the pCRII plasmid by digesting with SwaI and PacI and subsequently subcloned into SwaI/PacI digested pBANe6 to produce pBANe8 (FIG. 6).

pBANe8 was digested with PmeI and the linear PmeI fragment containing the NA2-tpi promoter, the lipase gene from Humicola lanuginosa, and the AMG terminator was isolated by preparative agarose electrophoresis using 40 mM Tris-acetate-1 mM disodium EDTA (TAE) buffer.

Aspergillus oryzae HowB430 was generated by transformation of Aspergillus oryzae HowB425 with the linear PmeI fragment according to the following procedure.

Aspergillus oryzae HowB425 was grown in 100 ml of 1% yeast extract-2% peptone-1% glucose at 32° C. for 16-18 hours with agitation at 150 rpm. The mycelia were recovered by filtration through a 0.45 μm filter until approximately 10 ml remained on the filter, washed with 25 ml of 1.0-1.2M MgSO₄ --10 mM sodium phosphate pH 6.5, filtered as before, washed again as before until 10 ml remained, and then resuspended in 10 ml of 5 mg/ml NOVOZYM 234™ (Novo Nordisk A/S, Bagsv.ae butted.rd, Denmark) in 1.2 M MgSO₄ --10 mM sodium phosphate pH 6.5 (0.45 μm filtered) in a 125 ml Ehrlenmeyer flask. The suspension was incubated with gentle agitation at 50 rpm for approximately one hour at 37° C. to generate protoplasts. A volume of 10 ml of the protoplast/mycelia preparation was added to a 30 ml Corex centrifuge tube, overlaid with 5 ml of 0.6 M sorbitol-10 mM Tris-HCl pH 7.5, and centrifuged at 3600×g for 15 minutes in a swinging bucket rotor to recover the protoplasts. The protoplasts were recovered from the buffer interface with a Pasteur pipet. The protoplasts were then washed with five volumes of STC, centrifuged, and then rewashed and centrifuged as before. The protoplasts were resuspended in STC to a final concentration of 2×10⁷ protoplasts per ml.

Transformation of Aspergillus oryzae HowB425 for amdS selection was conducted with protoplasts at a concentration of 2×10⁷ protoplasts per ml. Ten μg of DNA were added to 100 μl of protoplasts. A volume of 250 μl of PEG solution (60% PEG 4000-10 mM CaCl₂ --10 mM Tris-HCl pH 8.0) was then added and the mixture was placed at 37° C. for 30 minutes. Three ml of 1 M sorbitol-10 mM CaCl₂ --10 mM Tris pH 7.5 (STC) was added and the mixture was plated on Cove plates supplemented with 10 mM uridine selecting for amdS. The plates were incubated 7-10 days at 34° C. Transformants were transferred to plates of the same medium and incubated 3-5 days at 37° C. The transformants were purified by streaking spores and picking isolated colonies using the same plates of the same medium without sucrose under the same conditions.

Example 3

Construction of Aspergillus oryzae HowB427

Aspergillus oryzae HowB425 was co-transformed with pMHan37 and pSO2 (FIG. 7) to construct Aspergillus oryzae HowB427 to contain the lipase gene from Humicola lanuginosa behind the TAKA promoter.

pSO2 (FIG. 7) was constructed from a genomic library of Aspergillus oryzae 1560. The genomic library of Aspergillus oryzae 1560 was constructed by first partially digesting Aspergillus oryzae 1560 genomic DNA with Sau3A (New England Biolabs, Beverly, Mass.). Four units of Sau3A were used to digest 10 μg of Aspergillus oryzae 1560 genomic DNA using conditions recommended by the manufacturer. The reaction was carried out at 65° C., and samples were taken at 5 minute intervals (from 0 to 50 minutes). The reactions were placed on ice and stopped by the addition of EDTA to 10 mM. These digests were then run on a 1% agarose gel with ethidium bromide, and the region of the gel containing DNA from 3 kb to 9 kb was excised. The DNA was then purified from the gel slice using Beta-Agarase I using a protocol provided by the manufacturer (New England Biolabs, Beverly, Mass.). The size-selected DNA was then ligated into EMBL 4 arms according to the manufacturer's instructions (Clontech, Palo Alto, Calif.) at 16° C. overnight using conditions recommended by the manufacturer. The ligation reaction was packaged and titered using a Gigapack II Packaging Kit (Stratagene, La Jolla, Calif.) according to the manufacturer's protocol. A total of 16,000 recombinant plaques were obtained, and the library was amplified using a protocol provided by the manufacturer.

Appropriate dilutions of the genomic library were made to obtain 7000 plaques per 150 mm petri plate as described in the protocols provided with the EMBL 4 arms. The plaques were lifted to Hybond-N plus circular filters (Amersham, Cleveland, Ohio) using standard protocols (Sambrook et al., 1989, supra). The filters were fixed using UV crosslinking, and prehybridized at 42° C. (5×SSPE, 35% formamide). The genomic library was probed at low stringency (35% formamide, 5×SSPE at 42° C.) with a 500 bp fragment consisting of the Aspergillus niger pyrG gene which was labeled with ³² P using a random prime DNA labeling kit (Boehringer Mannheim, Indianapolis, Ind.). A 3.8 kb HindIII fragment was isolated from one phage and subcloned into a pUC118 cloning vector to produce pSO2.

The co-transformation of Aspergillus oryzae HowB425 was conducted using the procedure described in Example 2 except selection was on Minimal medium transformation plates. Transformants were transferred to Minimal medium transfer plates and incubated 3-5 days at 37° C. The transformants were then purified by streaking spores and picking isolated colonies using the same transfer plates under the same conditions.

Example 4

Construction of Plasmids pSO122, pDSY81, and pDSY82

pSO122 was constructed as described below to contain a 1.5 kb fragment of the Aspergillus oryzae pyrG gene.

PCR was used to generate pSO122 by introducing a BamHI restriction site at the 5' end of the pyrG gene of pSO2 using primers 7 and 8 shown below. Primers 7 and 8 were synthesized with an Applied Biosystems Model 394 DNA/RNA Synthesizer according to the manufacturer's instructions.

Primer 7: 5'-GCGGGATCCCTAGAGTAGGGGGTGGTGG-3' (SEQ ID NO:7)

Primer 8: 5'-GCCGGGATCCCCCCTAAGGATAGGCCCTA-3' (SEQ ID NO:8)

The amplification reaction (50 μl) contained the following components: 2 ng of pSO2, 48.4 pmoles of the forward primer, 48.4 pmoles of the reverse primer, 1 mM each of dATP, dCTP, dGTP, and dTTP, 1×Taq polymerase buffer, and 2.5 U of Taq polymerase (Perkin-Elmer Corp., Branchburg, N.J.). The reaction was incubated in an Ericomp Thermal Cycler programmed as follows: One cycle at 95° C. for 5 minutes followed by 30 cycles each at 95° C. for 1 minute, 55° C. for 1 minute and 72° C. for 2 minutes. The PCR product was isolated by electrophoresis on a 1% agarose gel.

The isolated PCR product was digested with BamHI and cloned into the BamHI site of pBluescript SK- (Stratagene, La Jolla, Calif.) to yield pS0122 (FIG. 8). The only homology between the genome of Aspergillus oryzae HowB430 and pS0122 was in the 5' end of the pyrG insert since the rest of the pyrG fragment was deleted from Aspergillus oryzae HowB430 as described in Example 2.

In order to reduce the frequency of targeting to this homologous region in the genome and since pSO122 contains two BamHI sites, two derivatives of pSO122, pDSY81 and pDSY82 (FIG. 8), were constructed in which one of the BamHI sites was destroyed. The plasmids pDSY81 and pDSY82 were constructed by partially digesting pSO122 with BamHI, filling-in the 5' overhangs with the Klenow fragment, closing down the plasmid by ligation and subsequent transformation into E. coli DH5α (Sambrook et al., 1989, supra). The transformants were then screened by extracting plasmid DNA from the transformants using a QIAwell-8 Plasmid Kit and restriction digesting the plasmid DNA with BamHI to determine if one of the BamHI sites had been destroyed. Plasmids with one of the BamHI sites destroyed were digested with NsiI/BamHI to determine which BamHI site had been destroyed.

Example 5

Aspergillus oryzae HowB430 transformation with pSO122, pDSY81, or pDSY82

Protoplasts of Aspergillus oryzae HowB430 were prepared as described in Example 2. A 5-15 μl aliquot of DNA (circular pSO122, pDSY81 linearized with 4 to 12 U of EcoRI, or pDSY82 linearized with 15 U of BamHI) was added to 0.1 ml of the protoplasts at a concentration of 2×10⁷ protoplasts per ml in a 14 ml Falcon polypropylene tube followed by 250 μl of 60% PEG 4000-10 mM CaCl₂ --10 mM Tris-HCl pH 7, gently mixed, and incubated at 37° C. for 30 minutes. The transformations were made either with 5 μg of circular pSO122, 6 μg of linearized pDSY81, or 6 μg of linearized pDSY82. Three ml of SPTC (1.2M sorbitol-10 mM CaCl₂ --10 mM Tris pH 8) were then added and the suspension was gently mixed. The suspension was mixed with 12 ml of molten overlay agar (1×COVE salts, 1% NZ amine, 0.8 M sucrose, 0.6% Noble agar) or 3 ml of STC medium and the suspension was poured onto a Minimal medium plate. The plates were incubated at 37° C. for 3-5 days.

The transformation frequencies of the circular pSO122 transformations ranged from about 100 to 200 transformants/μg. A library of ˜120,000 DNA-tagged transformants of Aspergillus oryzae HowB430 was obtained.

The transformation frequencies of the EcoRI REMI pDSY81 transformations ranged from about 60 to 100 per μg. An EcoRI REMI library of ˜28,000 DNA-tagged transformants of Aspergillus oryzae HowB430 was generated.

The transformation frequencies of the BamHI REMI pDSY82 transformations ranged from about 80 to 110 transformants/μg. A BamHI REMI library of ˜27,000 DNA-tagged transformants of Aspergillus oryzae HowB430 was obtained.

HindIII and Sa/I REMI libraries of Aspergillus oryzae HowB430 were also prepared using pDSY81 as described above.

The transformation frequencies of the HindIII REMI pDSY81 transformations ranged from about 80 to 120 per μg. A HindIII REMI library of 35,000 DNA-tagged transformants of Aspergillus oryzae HowB430 was generated.

The transformation frequencies of the Sall REMI pDSY81 transformations ranged from about 80 to 120 per μg. A SalI REMI library of 25,000 DNA-tagged transformants of Aspergillus oryzae HowB430 was generated.

The Aspergillus oryzae HowB430 tagged mutant library pools were designated "h" for pSO122; "e" for pDSY81 digested with EcoRI with subsequent transformation in the presence of EcoRI; "b" for pDSY82 digested with BamHI with subsequent transformation in the presence of BamHI; "hIII" for pDSY81 digested with HindIII with subsequent transformation in the presence of HindIII; and "s" for pDSY81 digested with SalI with subsequent transformation in the presence of Sall. There were 123 "h" pools, 28 "e" pools, 23 "b" pools, 55 "hII" pools, and 25 "s" pools.

The libraries described above were pooled into groups of ˜1000 transformants and stored in 10% glycerol at -80° C.

Example 6

Characterization of integration events in "REMI" Aspergillus oryzae HowB430 transformants

Genomic DNA was isolated from 26 of the EcoRI REMI transformants ("e" pool) described in Example 5 according to the following procedure. Each transformant was grown in 5 ml of YEG medium for 24 hours at 37° C. in a small Petri plate. Mycelia were then collected from each culture by filtration through Whatman filter paper No. 1 (Whatman, Springfield Mill, England) and transferred to a 1.7 ml centrifuge tube. The mycelia preparations were frozen in dry ice and dried in a SpeedVac (Savant Instruments, Inc., Farmingdale, N.Y.) overnight at room temperature. The frozen mycelia preparations were ground to a fine powder with a speared spatula and then the ground mycelia were resuspended in 0.5 ml of lysis buffer (100 mM EDTA, 10 mM Tris pH 8.0, 1% Triton X-100, 50 mM guanidine-HCl, 200 mM NaCl). RNase was added to each preparation to a final concentration of 20 μg/ml, and the preparations were incubated at 37° C. for 30 minutes. Protease K was then added to each preparation to a final concentration of 0.1 mg/ml, and the preparations were incubated at 50° C. for 1 hour. The preparations were centrifuged at 13,000×g for 15 minutes, and the supernatants were applied to QIAprep-8-well strips (Qiagen, Chatsworth, Calif.). The wells were washed once with 0.5 ml of PB and 0.75 ml of PE supplied by the manufacturer (Qiagen, Chatsworth, Calif.). After removing excess PE from each well, the DNAs were eluted from the wells in 200 μl of TE buffer (10 mM Tris-1 mM EDTA pH 7.0).

The genomic DNA was digested with either EcoRI to determine whether integration occurred into genomic EcoRI sites or SnaBI to determine whether or not the integration events were random throughout the genome by Southern hybridization according to the procedure described by Sambrook et al., 1989, supra. Southern blots of the digests were probed with a 1.6 kb NheI pyrG fragment obtained from pSO122 (FIG. 8) labeled with dioxygenin using a Genius Kit according to the manufacturer's instructions. The blot was prehybridized for 2 hours and hybridized overnight at 42° C. in DIG Easy Hyb. The blot was washed and processed as recommended by the manufacturer.

The Southern blot demonstrated that in 13 of 26 transformants, EcoRI linearized pDSY81 integrated into an EcoRI site in the genome, and the distribution of the integration events appeared to be random. In 20 of the 26 transformants, only a single copy of the plasmid was integrated while in 6 of the transformants at least 2 copies were integrated at the same locus. In order to determine if the bias (of 50% ) towards integration at EcoRI sites was due to REMI, genomic DNA was isolated as described above from 16 Aspergillus oryzae HowB425 transformants, in which the EcoRI enzyme was heat inactivated before transformation with EcoRI linearized pDSY81 according to the procedure described in Example 5, and submitted to Southern blot analysis as described above. Southern analysis of these transformants demonstrated that in none of the transformants did the plasmid integrate at an EcoRI site in the genome.

Example 7

Lipase Expression Screening

The Aspergillus oryzae HowB430 tagged mutant library "h", "e", and "b" pools described in Example 5 were assayed for lipase expression.

For 96-well plate screens, MY25 medium was diluted 1000-fold using a diluent made of equal volumes of sterile water and 2×MY Salts pH 6.5 solution. For 24-well plate methods, MY25 medium was diluted 100-fold using a diluent made of equal volumes of sterile water and 2×MY Salts pH 6.5 solution.

Primary 96-well plate screens involved the dilution of spores from distinct pools into MY25/1000 so that one spore on average was inoculated per well when 50 μl of medium was dispensed into the wells. After inoculation, the 96-well plates were grown for 7 days at 34° C. under static conditions. Cultures were then assayed for lipase activity as described below. Mutants of interest were inoculated directly into 24-well plates containing MY25/100 and were grown for 7 days at 34° C. Cultures were then assayed for lipase activity as described below. Mutants of interest were then plated on COVE plates to produce spores, spread on PDA plates to produce single colonies, and then 4 single colonies from each isolate were tested in the 24-well plate method described above.

The lipase assay substrate was prepared by diluting 1:50 a p-nitrophenylbutyrate stock substrate (21 μl of p-nitrophenylbutyrate/ml DMSO) into MC buffer (4 mM CaCl₂ --100 mM MOPS pH 7.5) immediately before use. Standard lipase (LIPOLASE™, Novo Nordisk A/S, Bagsv.ae butted.rd, Denmark) was prepared to contain 40 LU/ml of MC buffer containing 0.02% alpha olefin sulfonate (AOS) detergent. The standard was stored at 4° C. until use. Standard lipase was diluted 1/40 in MC buffer just before use. Broth samples were diluted in MC buffer containing 0.02% AOS detergent and 20 μl aliquots were dispensed to wells in 96-well plates followed by 200 μl of diluted substrate. Using a plate reader, the absorbance at 405 nm was recorded as the difference of two readings taken at approximately 1 minute intervals. Lipase units/ml (LU/ml) were calculated relative to the lipase standard.

The results of the 96-well screen followed by the 24-well screen identified for further evaluation 53 transformants from the pSO122 transformations and 44 transformants from the pDSY81 or pDSY82 REMI transformations. These identified transformants produced higher levels of lipase than the control strains Aspergillus oryzae HowB427 and Aspergillus oryzae HowB430.

Example 8

Shake flask and fermentation evaluation

The highest lipase-producing DNA-tagged mutants described in Example 7 were then plated onto COVE plates to produce spores for shake flask and fermentation evaluations.

Shake flask evaluations were performed by inoculating 300-500 μl of a spore suspension (0.02% Tween-80 plus spores from the COVE plates) into 25 ml of MY25 medium at pH 6.5 in a 125 ml shake flask. The shake flasks were incubated at 34° C. for 3 days at 200 rpm. Samples were taken at day 2 and day 3 and lipase activity was measured as described in Example 7.

The same DNA-tagged mutants were grown in a 2 liter lab fermentor containing medium composed of Nutriose, yeast extract, (NH₄)₂ HPO₄, MgSO₄ --7H₂ O, citric acid, K₂ SO₄, CaCl₂ --H₂ O, and trace metals solution at 34° C., pH 7, 1000-1200 rpm for 8 days. Lipase activity was measured as described in Example 7.

The results obtained are shown in Table 1 below where the lipase yield of either Aspergillus oryzae HowB427 or Aspergillus oryzae HowB430 as a control is normalized to 1.0.

                                      TABLE I     __________________________________________________________________________     Lipase Expression by DNA Tagged Mutants                               24-well                         # Screened                               Plate                                   Shake Flask                                         Ferm.     Strain              in 96-well                               Results                                   Results                                         Results     Description            Construction                      Pool                         Plates                               (LU/ml)                                   (LU/ml)                                         (LU/ml)     __________________________________________________________________________     HowB427            HowB425 + pMHan37                      NA NA    1.2 0.6   1.0     HowB430            HowB425 + pBANe8                      NA NA    1.0 1.0   NA     DEBY10.3            pDSY81 + BamHI                      b1 808   1.7 2.2   3.9     DEBY203.3            pDSY81 + EcoRI                      e1 707   2.4 2.1   1.8     DEBY599.3            pDSY81 + BamHI                      b18                         443   1.5 2.4   4.1     DEBY932            pDSY81 + EcoRI                      e21                         1092  1.9 1.9   3.6     DEBY1058            pDSY81 + BamHI                      b22                          80   2.3 2.4   3.8     DEBY1204.3.3            pDSY81 + EcoRI                      e26                         1260  1.9 2.0   3.0     HINL603            pDSY81 + HindIII                      hi3-7                         NA    2.8 2.0   3.3     HowL91.1            pSO122    h32                         1134  1.9 2.3   3.3     HowL214.2            pSO122    h9 861   1.9 2.3   3.0     HowL301.4            pSO122    h58                         731   2.3 2.8   3.6     HowL371.3            pSO122    h92                         592   1.8 2.5   3.5     HowL442.1            pSO122    h7 1095  2.3 2.6   4.5     HowL465.2            pSO122    h8 1003  2.3 2.2   3.2     HowL500.1            pSO122    h99                         885   2.3 2.7   3.4     HowL554.1            pSO122    h120                         892   1.9 2.3   3.6     HowL795.4            pSO122    h29                         1029  3.0 3.8   4.9     __________________________________________________________________________

As shown in Table I, the mutants produced approximately 2- to 4-fold more lipase than the control strain Aspergillus oryzae HowB427 and approximately 3- to 6-fold more lipase than the control strain Aspergillus oryzae HowB430 when grown in shake flasks. The mutants also produced approximately 2- to 5-fold more lipase than the control strain Aspergillus oryzae HowB427 when grown in fermentors.

Example 9

Rescue of Plasmid DNA and Flanking DNA from High Lipase Expressing Mutants

The plasmid DNA (pSO122, pDSY81, or pDSY82) and genomic flanking loci were isolated from mutants Aspergillus oryzae DEBY10.3, DEBY599.3, DEBY932, DEBY1058, DEBY1204.3.3, and HIN603.

Genomic DNA was isolated from mutants Aspergillus oryzae DEBY10.3, DEBY599.3, DEBY932, DEBY1058, DEBY1204.3.3, and HIN603 according to the following procedure. Spore stocks of each mutant were inoculated into 150 ml of YEG medium and were grown overnight at 37° C. and 250 rpm. The mycelia were harvested from each culture by filtration through Miracloth (Calbiochem, La Jolla, Calif.) and rinsed twice with TE. The mycelia preparations were then frozen quickly in liquid nitrogen and ground to a fine powder with a mortar and pestle. The powdered mycelia preparations were each transferred to a 50 ml tube and 20 ml of lysis buffer was added. RNAse was added to each preparation to a final concentration of 20 μg/ml, and the preparations was incubated at 37° C. for 30 minutes. Protease K was then added to each preparation to a final concentration of 0.1 mg/ml, and the preparations were incubated at 50° C. for 1 hour. The preparations were then centrifuged at 15,000×g for 20 minutes to pellet the insoluble material. Each supernatant was applied to a Qiagen MAXI column (Qiagen, Chatsworth, Calif.) which was equilibrated with QBT provided by the manufacturer. The columns were then washed with 30 ml of QC provided by the manufacturer. DNA was eluted from each column with 15 ml of QF provided by the manufacturer and then recovered by precipitation with a 0.7 volume of isopropanol and centrifugation at 15,000×g for 20 minutes. The pellets were finally washed with 5 μl of 70% ethanol, air-dried, and dissolved in 200 μl of TE.

Two μg aliquots from each of the Aspergillus oryzae DEBY1.03, DEBY599.3, DEBY932, DEBY1058, DEBY1204.3.3, and HIN603 genomic DNA preparations were digested separately with BglII HpaI, NarI, NdeI, SphI, and StuI. The restriction endonucleases did not cut pDSY82 which allowed the isolation of the integrated plasmid and the flanking genomic DNA. The digested genomic DNAs were then ligated in a 20 μl reaction with T4 DNA ligase.

The ligated DNA preparations were each transformed into E. coli HB101 or E. coli DH5α. The transformants were then screened by extracting plasmid DNA from the transformants, restriction digesting the inserts to confirm they are derived from pDSY82, and sequencing the inserts according to the method described above using primers specific to pDSY82.

Transformant E. coli HB101-pDSY112 contained the HpaI rescued locus from mutant Aspergillus oryzae DEBY599.3. Transformant E. coli HB101-pDSY109 contained the SphI rescued locus from mutant Aspergillus oryzae DEBY10.3. Transformant E. coli HB101-pDSY138 contained the NdeI rescued locus from mutant DEBY932. Transformant E. coli HB101-pDSY141 contained the BglII rescued locus from mutant DEBY1058. Transformant E. coli DH5α-pSMO1204 contained the BgllI rescued locus from mutant Aspergillus oryzae DEBY1204.3.3. Transformant E. coli DH5α-pSMOH603 contained the BglII rescued locus from mutant Aspergillus oryzae HIN603.

Example 10

Characterization of Aspergillus Oryzae DEBY599.3 Rescued Locus pDSY112

The Aspergillus oryzae DEBY599.3 rescued locus pDSY112 containing 1625 bp was sequenced according to the method described in Example 2. The nucleic acid sequence (SEQ ID NO:9) and the deduced amino acid sequence (SEQ ID NO:10) are shown in FIG. 9. The nucleic acid sequence suggested that integration occurred within the promoter of a glucose transporter about 150 bp upstream of the ATG start codon. The open reading frame was punctuated by an intron. The predicted protein (SEQ ID NO:10) shared 31.6% and 24.8% identity with the glucose transporters from yeast (SEQ ID NO:11) and human (SEQ ID NO:12), respectively, and 20.1% identity with an inositol transporter from yeast (SEQ ID NO:13). Glucose transporters have very distinct predicted secondary structures with 12 membrane spanning domains. Kyte-Doolittle plots of the Aspergillus oryzae DEBY599.3 rescued locus predicted 12 membrane spanning domains similar to the yeast and human glucose transporters.

In order to confirm that the rescued flanking DNA was the gene disrupted in Aspergillus oryzae DEBY599.3, a Southern blot of Aspergillus oryzae HowB101 and Aspergillus oryzae DEBY599.3 genomic DNA preparations digested with BgllI was prepared and analyzed according to the procedure described in Example 6. The blot was probed with the Aspergillus oryzae DEBY599.3 rescued flanking DNA at 42° C. in DIG Easy Hyb. The blot was then washed and processed using protocols provided with a Genius Kit.

A BgIII band of 2.7 kb from Aspergillus oryzae HowB101 hybridized with the probe, while an ˜8 kb BglII band from Aspergillus oryzae DEBY599.3 hybridized to the probe. The size difference corresponded to the length of the plasmid integrated during REMI confirming the DNA rescued from Aspergillus oryzae DEBY599.3 was flanking the insertion.

Example 11

Aspergillus oryzae transformation with HpaI linearized pDSY112 and lipase expression screening

Aspergillus oryzae HowB430 was transformed with HpaI digested pDSY112 and the transformants were recovered using the methods described in Example 5. Totally, 216 transformants were grown in 24 well microtiter plates in 1/100 strength MY25 medium. Samples were taken at 4 and 6 days and assayed for lipase activity as described in Example 7. An equal number of low, average and high producing lipase transformants were spore purified and retested in 24 well microtiter cultures as described above. These purified transformants were also tested in shake flasks in full-strength MY25 medium as described in Example 8. The top five producing transformants were then grown in a 2 liter fermentor as described in Example 8. Lipase activity was measured as described in Example 7.

The results obtained are shown in Table 2 below where the lipase yield of Aspergillus oryzae HowB430 is normalized to 1.0.

                  TABLE 2     ______________________________________     Strain     Fermentation Results (Relative LU/ml)     ______________________________________     HowB430    1.0     DEBY599.3  1.7     112T90.2.2 2.3     112T100.4.2                2.1     112T344.2.1                1.7     112T142.2  2.0     112T59.2   2.4     ______________________________________

All five retransformants produced approximately the same level of lipase activity as the original tagged strain Aspergillus oryzae DEBY599.3 when grown under fermentation conditions. In order to determine if the pDSY112 had integrated at the same homologous locus in the genome, a Southern blot of Aspergillus oryzae HowB430, Aspergillus oryzae DEBY599.3 and the pDSY112 transformants genomic DNA preparations digested with BglII was prepared and analyzed according to the procedure described in Example 6. The blot was probed with the Aspergillus oryzae DEBY599.3 rescued flanking DNA at 42° C. in DIG Easy Hyb. The blot was washed and processed using protocols provided with a Genius Kit.

A BglII band of 2.7 kb from Aspergillus oryzae HowB430 hybridized with the probe, while an ˜8 kb BglII band from Aspergillus oryzae DEBY599.3 hybridized to the probe. A wild-type BglII band of 2.7 kb and a second band corresponding to the transforming DNA hybridized to the probe in all of the transformants. Therefore, none of the retransformants had exact gene replacements.

Example 12

Characterization of Aspergillus oryzae DEBY10.3 Rescued Locus pDSY109

The 3.4 and 2.2 kb regions on either side of the integration event of the Aspergillus oryzae DEBY10.3 rescued locus pDSY109 were sequenced according to the procedure described in Example 2. The nucleic acid sequence suggested that the integration event occurred within the open reading frame of a palB gene. palB genes encode a cysteine protease involved in the signal transduction pathway that signals ambient pH.

The genomic library of Aspergillus oryzae HowB430 was constructed by first partially digesting Aspergillus oryzae HowB430 genomic DNA with Tsp509I. Four units of Tsp509 were used to digest 3.5 μg of Aspergillus oryzae HowB430 genomic DNA using conditions recommended by the manufacturer. The reaction was carried out at 65° C., and samples were taken at 5 minute intervals (from 0 to 50 minutes). The reactions were placed on ice and stopped by the addition of EDTA to 10 mM. These digests were then run on a 1% agarose gel with ethidium bromide, and the region of the gel containing DNA from 3 kb to 9 kb was excised. The DNA was then purified from the gel slice using Beta-Agarase I using a protocol provided by the manufacturer (New England Biolabs, Beverly, Mass.). The size-selected DNA was then ligated into Lambda ZipLox EcoRI arms according to the manufacturer's instructions at 16° C. overnight using conditions recommended by the manufacturer. The ligation reaction was packaged and titered using a Gigapack GoldIII Packaging Kit according to the manufacturer's protocol. 8×10⁶ recombinant plaques were obtained, and the library was amplified using a protocol provided by the manufacturer.

The genomic library was screened to obtain a genomic clone of palB. Appropriate dilutions of the genomic library were made to obtain 7000 plaques per 150 mm petri plate as described in the protocols provided with the Lambda ZipLox arms. The plaques were lifted to Hybond-N plus circular filters using standard protocols (Sambrook et al., 1989, supra). The filters were fixed using UV crosslinking, and prehybridized at 42° C. in DIG Easy Hyb. The filters were hybridized with a DIG-labeled 0.25 kb palB probe. The probe was labeled with dioxygenin using a Genius Kit and PCR amplified with the following primers synthesized with an Applied Biosystems Model 394 DNA/RNA Synthesizer according to the manufacturer's instructions:

5'-CTGCCGTCGAAGGTGTCCAAG-3' (SEQ ID NO: 14)

5'-ATTGTGGCCCCTATGTGGATT-3' (SEQ ID NO:15)

The parameters for PCR are as described in Example 2. The filters were washed and processed post-hybridization using protocols provided with the Genius Kit. Several positive plaques were identified and purified to homogeneity using standard protocols (Sambrook et al., 1989, supra).

The nucleotide sequence was determined for the palB gene according to the method described in Example 2. The nucleic acid sequence (SEQ ID NO:16) and the deduced amino acid sequence (SEQ ID NO:17) are shown in FIG. 10. The open reading frame was interrupted by 3 introns. The Aspergillus oryzae PalB protein (SEQ ID NO:17) shared 66.4% identity with the Aspergillus nidulans PalB protein (SEQ ID NO:18). The site of insertion also contained a highly conserved domain of 37 amino acids (SEQ ID NO:19) similar to that derived from the Neurospora crassa NADH dehydrogenase (SEQ ID NO:20) which was probably a piece of mitochondrial DNA that inserted during transformation or rescue in E. coli.

A Southern blot of Aspergillus oryzae DEBY10.3 and Aspergillus oryzae HowB101 genomic DNA digested with BgllI was prepared according to the method described in Example 6. The blot was probed with the Aspergillus oryzae DEBY10.3 rescued flanking DNA to confirm that the rescued flanking DNA was the gene disrupted in Aspergillus oryzae DEBY10.3.

A BglII band of ˜7.5 kb from Aspergillus oryzae HowB101 hybridized to the probe while a band of 12 kb from Aspergillus oryzae DEBY10.3 hybridized to the probe. The size difference was the expected size for one plasmid copy being integrated confirming the locus rescued was disrupted in Aspergillus oryzae DEBY10.3.

Because the integration event in Aspergillus oryzae DEBY10.3 would be predicted to lead to a nonfunctional PalB protein, Aspergillus oryzae DEBY10.3 was tested for growth at pH 8.0 and pH 6.5. Aspergillus nidulans palB minus strains are unable to grow at pH 8.0 but are able to grow at pH 6.5. Aspergillus oryzae HowB430 and Aspergillus oryzae DEBY10.3 were grown in Minimal medium with 10 mM uridine at either pH 8.0 or pH 6.5. As predicted, Aspergillus oryzae DEBY10.3 was unable to grow at pH 8.0.

Example 13

Construction of pMT1936

pMT1936 was constructed to contain a disruption cassette of palB using the following primers synthesized with an Applied Biosystems Model 394 DNA/RNA Synthesizer according to the manufacturer's instructions.

100752: 5'-GGTTGCATGCTCTAGACTTCGTCACCTTATTAGCCC-3' (SEQ ID NO:21)

100753: 5'-TTCGCGCGCATCAGTCTCGAGATCGTGTGTCGCGAGTACG-3' (SEQ ID NO:22)

100754: 5'-GATCTCGAGACTAGTGCGCGCGAACAGACATCACAGGAACC-3' (SEQ ID NO:23)

100755: 5'-CAACATATGCGGCCGCGAATTCACTTCATTCCCACTGCGTGG-3' (SEQ ID NO:24)

The Aspergillus oryzae palB 5' flanking sequence and the sequence encoding the N-terminal part of the palB product were PCR amplified from genomic DNA of Aspergillus oryzae A1560 obtained according to the method described in Example 2. Approximately 0.05 μg of DNA template and 5 pmole of each of the two primers 100755 and 100754 were used. Amplification was performed with the polymerase Pwo as described by the manufacturer (Boehringer Mannheim, Indianapolis, Ind.). Amplification proceeded through 40 cycles. Part of the reaction product was phenol extracted, ethanol precipitated, digested with restriction enzymes EcoRI and XhoI and a fragment of approximately 1.05 kb was isolated by agarose gel electrophoresis.

The Aspergillus oryzae palB 3' flanking sequence and the sequence encoding the C-terminal part of the palB gene product were obtained as described above except that primers 100753 and 100752 were used for amplification and the PCR product was digested with restriction enzymes XhoI and XbaI before gel electrophoresis to recover a fragment of approximately 1.50 kb.

The two digested and purified PCR fragments described above were ligated in a three part ligation with the purified 2.7 kb EcoRI-XbaI fragment from the vector pJaL400 (FIG. 11) to produce pMT1935 (FIG. 12). The palB 5' and 3' flanks of pMT1935 are separated by BssHII, SpeI, and XhoI sites introduced via PCR primers 100754 and 100753.

To insert an Aspergillus oryzae pyrG gene between the palB 5' flank and the 3' flank of pMT1935, the 3.5 kb HindIII fragment of pJaL394 (FIG. 13) containing the repeat flanked pyrG gene was cloned into HindIII cut, dephosphorylated and purified pBluescript II SK (-). Plasmids with inserts in either orientation were obtained. One plasmid, pMT1931 (FIG. 14), was selected in which the SpeI site of the pBluescript polylinker was downstream of the pyrG gene and the XhoI site was upstream of the pyrG gene. The pyrG gene was isolated as a 3.5 kb SpeI-XhoI fragment and inserted in SpeI and XhoI digested and purified pMT1935 to produce the disruption plasmid pMT1936 (FIG. 15).

The pyrG selectable palB disruption cassette can be isolated from pMT1936 as a 6.2 kb NotI fragment (NotI cutting in polylinkers) or as a 5.5 kb AseI-PvuI fragment (AseI and PvuI cutting within the actual palB 5' and 3' flanking sequences).

Example 14

Aspergillus oryzae transformation with AseI/PvuI palB disruption cassette from pMT1936 and lipase screening

Aspergillus oryzae HowB430 was transformed using the same transformation procedure described in Example 5 with a 5.5 kb AseI/PvuI fragment obtained from pMT1936. The linear fragment for transformation was isolated by digestion of pMT1936 with AseI and PvuI and separation of the fragment on a 1% agarose gel using a QIAquick Gel Extraction Kit according to the manufacturer's instructions. The transformants were then tested for growth on Minimal medium plates at pH 6.5 or pH 8.0.

The results showed that 13 of the 128 transformants tested possessed the palB minus phenotype as indicated by the inability to grow at pH 8.0. The 13 palB minus strains and 13 of the transformants that were able to grow at pH 8.0 were spore purified and then evaluated in 24-well plate and shake flask cultures for lipase production using the methods described in Examples 7 and 8, respectively. The results are shown in Table 3 below.

Southern blots of the genomic DNA from an Aspergillus oryzae palB minus mutant, an Aspergillus oryzae palB plus strain, and Aspergillus oryzae HowB430 were performed to determine if the AsnI/PvuI transforming DNA fragment had integrated as a clean replacement into the palB locus. The genomic DNAs were prepared according to the procedure described in Example 9, digested with PvuI, and electrophoresed on a 0.8% agarose gel. The DNAs were transferred to a Hybond N⁺ membrane using 0.4 N NaOH and capillary action. The blot was UV crosslinked prior to prehybridization at 65° C. in Rapid Hyb. The blot was then probed with a 0.9 kb AsnI/SpeI fragment from pMT1936. The 0.9 kb fragment was isolated from an agarose gel slice using QiaQuick spin column after electrophoreses on a 1% agarose gel. The fragment was labeled using Vistra ECF Random Prime Labeling Kit. The blots were prehybridized and hybridized at 65° C. in Rapid Hyb (Amersham, Cleveland, Ohio), and then washed twice for 5 minutes in 2×SSC, 0.1% SDS at 65° C. and twice for 10 minutes in 0.2×SSC, 0. 1% SDS at 65° C. Following the washes, the blot was processed for detection using the Vistra ECF Signal Amplification Kit (Amersham, Cleveland, Ohio) and the STORM860 Imaging System (Molecular Dynamics, Sunnyvale, Calif.).

The Southern blot results demonstrated that the probe hybridized to a band of 6 kb from Aspergillus oryzae HowB430. A clean disruption would be expected to hybridize to about an 8 kb PvuI band. The Southern blot results further showed that some of the palB minus strains had clean disruptions while others did not. The Southern blot results are summarized in Table 3.

Three of the palB minus strains were also run under fermentation conditions according to the procedure described in Example 8. The results obtained are shown in Table 3 below where the lipase yield of Aspergillus oryzae HowB430 is normalized to 1.0. The three palB minus strains performed better or close to the same as the original tagged mutant Aspergillus oryzae DEBY10.3.

                  TABLE 3     ______________________________________                                     Fermentation            PalB     24 well Shake flasks                                     results Southern     Strain phenotype                     LU/ml   LU/ml   LU/ml   pattern     ______________________________________     HowB430            plus     1.0     1.0     1.0     wild type     palB3-1            plus     1.2     1.1     1.7     wild-type                                             and other     palB4-1            plus     1.0     0.8     1.4     wild-type                                             and other     palB5-1            minus    1.4     1.4     2.0     disrupted     palB8-1            plus     0.9     1.0     NA      wild-type                                             and other     palB18-1            plus     0.9     NA      NA      wild-type                                             and other     palB27-1            plus     1.0     NA      NA      wild-type                                             and other     palB29-1            minus    0.8     NA      NA      other     palB30-1            plus     0.9     NA      1.0     wild-type                                             and other     palB31-1            minus    1.3     NA      NA      other     palB37-1            plus     0.8     NA      NA      wild-type                                             and other     palB39-1            plus     0.9     NA      NA      wild-type                                             and other     palB41-1            plus     1.0     0.8     NA      wild-type                                             and other     palB42-1            plus     1.2     1.0     NA      wild-type                                             and other     palB43-1            minus    1.3     1.2     NA      other     palB69-1            plus     1.2     1.4     NA      wild-type                                             and other     palB71-1            minus    1.2     1.3     1.8     other     palB72-1            minus    1.5     1.6     2.0     other     palB75-1            minus    1.3     1.4     1.3     other     palB76-1            minus    1.6     1.3     2.0     clean                                             disruption     palB79-1            plus     1.2     1.0     NA      wild-type                                             and other     ______________________________________

Example 15

Characterization of Aspergillus oryzae DEBY932 Rescued Locus pDSY138

The Aspergillus oryzae DEBY932 rescued locus pDSY138 containing 1625 bp was sequenced according to the method described in Example 2. The nucleic acid sequence (SEQ ID NO:25) and deduced amino acid sequence (SEQ ID NO:26) are shown in FIG. 16. The nucleic acid sequence showed that the EcoRI site of the REMI integration was 810 bp upstream of the ATG start codon for an open reading frame and the deduced amino acid sequence (SEQ ID NO:26) had significant identity to mannitol-1-phosphate dehydrogenases from E. coli and Bacillus subtilis. The open reading frame coded for a predicted protein of 319 amino acids, and shared 13.3% and 34.7% identity with the E. coli (SEQ ID NO:27) and the Bacillus subtilis (SEQ ID NO:28) mannitol-1-phosphate dehydrogenases, respectively.

A Southern blot of Aspergillus oryzae DEBY932 and Aspergillus oryzae HowB430 genomic DNA preparations digested with NdeI was prepared and analyzed according to the method described in Example 14. The blot was probed with the Aspergillus oryzae DEBY932 rescued flanking DNA to confirm that the rescued flanking DNA is the gene disrupted in DEBY932.

An NdeI band of approximately 5 kb from Aspergillus oryzae HowB430 hybridized to the rescued locus while a band of approximately 10 kb from Aspergillus oryzae DEBY932 hybridized to the probe confirming that the rescued locus was the disrupted locus in Aspergillus oryzae DEBY932.

Example 16

Aspergillus oryzae Transformation with NdeI Linearized pDSY138 and Lipase Expression Screening

Aspergillus oryzae HowB430 was transformed with NdeI digested pDSY138 and the transformants were recovered using the methods described in Example 5. Totally, 180 recovered transformants were grown in 24 well microtiter plates in 1/100 strength MY25, and samples were taken at 4 and 6 days for lipase assays as described in Example 7. The top 11 highest lipase producing and 1 average lipase producing transformants were spore purified and retested in 24 well microtiter cultures. These purified transformants were also evaluated in shake flasks in full-strength MY25 as described in Example 8. The top two producers were also grown in a 2 liter fennentor as described in Example 8. Lipase activity was measured as described in Example 7.

The results obtained are shown in Table 4 below where the lipase yield of Aspergillus oryzae HowB430 was normalized to 1.0. The top two lipase producers produced essentially the same amount of lipase activity as the original tagged mutant Aspergillus oryzae DEBY932.

                  TABLE 4     ______________________________________                 Fermentation Results     Strain      (Relative LU/ml)                               Southern Results     ______________________________________     HowB430     1.0           Wild-type     DEBY932.3.3 2.1           Disrupted     138T83.1.1  2.2           Disrupted     138T102.1.1 1.9           Disrupted     ______________________________________

A Southern blot of Aspergillus oryzae DEBY932, Aspergillus oryzae HowB430 and pDSY138 genomic DNA preparations digested with NdeI was prepared and analyzed as described in Example 14 to determine if pDSY138 had integrated at the homologous locus producing gene replacements in the transformants using the Aspergillus oryzae DEBY932 rescued flanking DNA as a probe.

The Southern blot showed that an NdeI band of approximately 5 kb from Aspergillus oryzae HowB430 hybridized to the rescued locus while a band of approximately 10 kb from Aspergillus oryzae DEBY932 hybridized to the probe. In Table 4, the column labeled Southern results indicated whether the transformants had a wild-type NdeI fragment of the size observed in the parent strain Aspergillus oryzae HowB430 or whether the transformants had a band corresponding to the disrupted size observed in Aspergillus oryzae DEBY932.

Example 17

Characterization of Aspergillus oryzae DEBY1058 Rescued Locus pDSY141

The Aspergillus oryzae DEBY1058 rescued locus pDSY141 containing approximately 1 kb was sequenced according to the method described in Example 2. The nucleic acid sequence demonstrated that the rescued locus contained flanking DNA from only one side of the BamHI REMI integration event, and the pDSY141 sequence had rearranged.

A Southern blot of Aspergillus oryzae DEBY 1058 genomic DNA digested with BamHI was probed with the Aspergillus oryzae DEBY1058 rescued flanking DNA was prepared and analyzed as described in Example 14 to confirm that the rescued flanking DNA is the gene disrupted in Aspergillus oryzae DEBY1058.

The Southern analysis showed that the pDSY82 DNA had integrated as a REMI event at a BamHI site, but more than one copy of pDSY82 had integrated which suggested why the rescued plasmid had rearranged and only contained one side of the flanking DNA.

In order to obtain the other flanking piece, a genomic clone (pDSY163) was isolated from the Aspergillus oryzae HowB430 genomic library, prepared as described in Example 6, using a ³² P-labeled 0.5 kb fragment of the rescued genomic DNA from Aspergillus oryzae DEBY1058. The probe was labeled using a Prime-It Kit according to the manufacturer's instructions (Stratagene, La Jolla, Calif.). Five plates of approximately 7000 plaques each were plated, and the plaques were lifted to Hybond-N⁺ as described in Example 12. The filters were prehybridized at 42° C. in 50% formamide, 5×SSPE, 0.3% SDS and 200 μg/ml of sheared and denatured salmon sperm DNA for 1 hour. The denatured probe was added, and the filters were hybridized overnight at 42° C. The filters were washed in 1×SSC, 0.1% SDS for 5 minutes at 65° C. twice, in 0. 1×SSC, 0. 1% SDS at 65° C. for 15 minutes twice, and in 2×SSC at room temperature for 10 minutes. The filters were exposed to X-ray film, and 12 positive plaques were picked and purified using standard protocols (Sambrook et al., 1989, supra). Plasmid DNA was isolated from the purified genomic clones using the excision protocol provided with the Lambda ZipLox EcoRI Arms Kit.

The nucleotide sequence of 3.6 kb of the genomic clone was determined as described in Example 2. The nucleic acid sequence (SEQ ID NO:29) and deduced amino acid sequence (SEQ ID NO:30) are shown in FIG. 17. The nucleic acid sequence showed that the BamHI site of integration in the mutant is 250 bp downstream of the stop codon for an open reading frame that encodes a protein (SEQ ID NO:30) which shared significant identity with manganese superoxide dismutase from Saccharomyces cerevisiae (SEQ ID NO:31).

Since the site of integration in Aspergillus oryzae DEBY1058 was 250 bp downstream of the stop codon for the manganese superoxide dismutase gene, the effect of this integration on expression of the manganese superoxide dismutase was determined. Saccharomyces cerevisiae strains lacking a functional manganese superoxide dismutase are sensitive to paraquat when grown in the presence of oxygen. Aspergillus oryzae DEBY1058 and Aspergillus oryzae HowB430 were grown in 24 well microtiter plates in 1 ml of YEG medium supplemented with 10 mM uridine and either 0, 2, 4, 6, 8, 10 or 20 mM paraquat at 34° C. with shaking. Aspergillus oryzae HowB430 grew at concentrations of paraquat up to 8 mM while growth of Aspergillus oryzae DEBY1058 was inhibited by 2 mM paraquat. The data indicated that the integration event 250 bp downstream of the stop codon for manganese superoxide dismutase in Aspergillus oryzae DEBY1058 reduced expression of manganese superoxide dismutase.

Example 18

Construction of pDSY162

pDSY162 was constructed to contain a disruption cassette for manganese superoxide dismutase by PCR amplification of a 3179 bp XbaI/KpnI fragment of genomic DNA containing the manganese superoxide dismutase gene using the following primers synthesized with an Applied Biosystems Model 394 DNA/RNA Synthesizer according to the manufacturer's instructions.

970738: 5'-GCTCTAGATCGTCGGAGCTCATGTCGGCGATTTTAC-3' (SEQ ID NO:32)

970739: 5'-GCGGTACCACGCCTAGAGCAAAGTATAAATAAGGAA-3' (SEQ ID NO:33)

The amplification reaction (100 μl) contained the following components: 0.2 μg of the pDSY163, 48.4 pmol of primer 970738, 48.4 pmol of primer 979739, 1 mM each of dATP, dCTP, dGTP, and dTTP, 1×Taq polymerase buffer, and 2.5 U of Taq polymerase. The reaction was incubated in an Ericomp Thermal Cycler programmed as follows: One cycle at 95° C. for 5 minutes followed by 30 cycles each at 95° C. for 1 minute, 55° C. minute, and 72° C. for 2 minutes. Two μl of the reaction were electrophoresed on an agarose gel to confirm the amplification of the PCR product of approximately 3179 bp.

The PCR product was subcloned into pCR®TOPO using a TOPO TA Cloning Kit (Invitrogen, San Diego, Calif.). The transformants were then screened by extracting plasmid DNA from the transformants using a QIAwell-8 Plasmid Kit according to the manufacturer's instructions, restriction digesting the plasmid DNA using XbaI/KpnI to confirm the presence of the correct size fragment, and sequencing the DNA according to the method described in Example 2 to confirm the PCR product.

The plasmids containing the manganese superoxide dismutase insert were digested with XbaI and KpnI and separated on a 1% agarose gel. A 3.1 kb manganese superoxide dismutase fragment was purified using a QIAquick Gel Extraction Kit according to the manufacturer's instructions. The purified fragment was ligated with pBluescript SK- digested with XbaI and KpnI to produce pDSY161 (FIG. 18). The ligation reaction was used to transform E. coli DH5α.

The transformants were then screened by extracting plasmid DNA from the transformants using a QIAwell-8 Plasmid Kit according to the manufacturer's instructions and digesting the plasmids with HindIII to determine which clones were correct.

pDSY161 was digested with HindIII to remove a 600 bp fragment, and the digestion was electrophoresed on a 1% agarose gel. A 5.4 kb vector fragment was isolated using a QIAquick Gel Extraction Kit according to the manufacturer's instructions, and ligated to the 3.5 kb HindIII fragment from pJaL394 (FIG. 13) containing a pyrG gene repeat to produce pDSY162 (FIG. 19). The ligation reaction was used to transform E. coli DH5α.

The transformants were then screened by extracting plasmid DNA from the transformants using a QIAwell-8 Plasmid Kit according to the manufacturer's instructions and digesting them with HindIII to determine which plasmids contained the expected 3.5 kb HindIII fragment in pDSY162.

Example 19

Aspergillus oryzae Transformation with AsnI/PvuI Manganese Superoxide Dismutase Disruption Cassette and Lipase Screening

Aspergillus oryzae HowB430 was transformed with a 5.8 kb AseI/PvuI fragment containing the manganese superoxide dismutase disruption cassette using the same transformation procedure described in Example 5. The linear fragment for transformation was isolated by digestion of pDSY162 with AseI and PvuI and separation of the fragment on a 1% agarose gel using a QIAquick Gel Extraction Kit according to the manufacturer's instructions. The transformants were then tested for growth on Minimal medium plates at pH 6.5.

Six transformants were obtained and were tested for sensitivity to paraquat as described in Example 17. Four of the 6 transformants were paraquat sensitive indicative of the manganese superoxide dismutase disruption minus phenotype although the four paraquat sensitive strains were not equally sensitive to paraquat. As shown in Table 5 below, a dash means not sensitive to paraquat, ++ means sensitive means sensitive to intermediate levels of paraquat and +++ means inhibited by even 2 mM paraquat. All of the transformants were spore purified and tested in 24 well and shake flask cultures for lipase production according to the procedures described in Examples 7 and 8. The results tabulated in Table 5 below show that following transformation of Aspergillus oryzae HowB430 with the manganese superoxide dismutase disruption cassette, transformants sensitive to paraquat on average produced higher LIPOLASE™ levels than Aspergillus oryzae HowB430.

Southern blots of the genomic DNA from an Aspergillus oryzae manganese superoxide dismutase minus mutant, an Aspergillus oryzae manganese superoxide dismutase plus strain, and Aspergillus oryzae HowB430 were performed as described in Example 14 to determine if the AsnI/PvuI transforming DNA fragment had integrated as a clean replacement into the manganese superoxide dismutase locus.

The results of the Southern blot (Table 5) showed that strains sensitive to even 2 mM paraquat were disrupted at the manganese superoxide dismutase locus while those sensitive to intermediate levels of paraquat have both a wild-type locus and the disrupted cassette locus. The Souther blot and LIPOLASE™ yield results together suggests that expression of both full length and truncated manganese superoxide dismutase in the same cell leads to an intermediate sensitivity to paraquat and an increase in LIPOLASE™ production. This can be explained by the fact that manganese superoxide dismutase is a homodimer so expression of the wild-type and truncated forms coded for by the wild-type and disrupted cassette, respectively, leads to heterodimers which are either non-functional or partially functional.

                  TABLE 5     ______________________________________                      24 well   Shake flasks                      results   results             Paraquat (Relative (Relative     Strain  sensitivity                      LU/ml)    LU/ml)  Southern Results     ______________________________________     HowB430 -        1.0       1.0     wild-type     430162T1             -        1.0       1.1     wild-type & other     430162T2             ++       1.3       2.5     wild-type & other     430162T3             ++       1.4       3.0     wild-type & other     430162T4             +++      1.2       2.1     disrupted     430162T5             +++      1.0       1.5     disrupted     430162T6             -        0.9       1.2     wild-type & other     ______________________________________

Example 20

Characterization of Aspergillus oryzae DEBY1204.3.3 Rescued Locus pSMO1204

The Aspergillus oryzae DEBY1204.3.3 rescued locus pSMO1204 containing 2.0 kb was sequenced according to the procedure described in Example 2. The nucleic acid sequence (SEQ ID NO:34) as shown in FIG. 20 had no sequence homology to any published sequences.

Southern analysis and sequencing of a genomic clone was used to confirm that no deletions had taken place when the tagged mutant was generated. A Southern blot of Aspergillus oryzae HowB430 genomic DNA digested with various restriction endonucleases (BamHI, BgllI, Sall and SphI) was prepared and analyzed as described in Example 14. Probes from both ends of the rescued plasmid were generated by PCR using the primers described below. The primers were synthesized with an Applied Biosystems Model 394 DNA/RNA Synthesizer according to the manufacturer's instructions.

970052 5'-CTATGATTGGCCGATAGG-3' (SEQ ID NO:35)

970053 5'-CCAGGCTCGCACGCTTTC-3' (SEQ ID NO:36)

970054 5'-CTTGCAACTAACGGGGTT-3' (SEQ ID NO:37)

970055 5'-TGAGAAAGACCAAGAATG-3' (SEQ ID NO:38)

Probe 1 was generated from one end of the rescued locus by PCR using primer 970052 and primer 970053. Probe 2 was generated from the other end of the rescued locus by PCR using primer 970054 and primer 970055. The amplification/labeling reaction (50 μl) contained the following components: 10 ng rescued plasmid pSMO1204, 50 pmole each of primer 970052 and 970053 for probe 1 or 50 pmole each of primer 970054 and primer 970055 for probe 2, 1×DIG labeling mix (Boehringer Mannheim, Indianapolis, Ind.), 1×Taq polymerase buffer, and 2.5 U of Taq polymerase. The reaction was incubated in an Ericomp Thermal Cycler programmed as follows: One cycle at 95° C. for 5 minutes followed by 30 cycles each at 95° C. for 1 minute, 58° C. for 1 minute, and 72° C. for 1.5 minutes

Southern blots of Aspergillus oryzae HowB430 genomic DNA digested with various restriction enzymes were prepared and analyzed according to the procedure described in Example 14. The blots were hybridized independently to probe 1 and probe 2. Identical banding patterns of the digests with both probes would suggest that a deletion had not occurred. Conditions of the Southern analysis were as follows: Blots were prehybridized for 1 hour and hybridized overnight at 42° C. in Easy Hyb. Blots were washed in 2×SSC, 0.1% SDS twice at room temperature for 15 minutes each, then washed twice at 65° C. in 0.1×SSC, 0.1% SDS for 15 minutes each. Detection continued with buffers and reagents from Boehringer Mannheim's DIG Wash Block Buffer System. CDP star (Boehringer Mannheim, Indianapolis, Ind.) was used to detect the chemiluminescent reaction. Film was exposed for approximately 1 hour.

The Southern blot results showed identical banding patterns with the different digests suggesting a direct insertion of the tagged plasmid. A genomic clone was obtained by probing an Aspergillus oryzae HowB430 Ziplox library obtained as described in Example 12 with the probe from the tagged mutant Aspergillus oryzae DEBY1204. The Ziplox library of Aspergillus oryzae HowB430 was screened with probe 1. A genomic clone was isolated and sequenced (Example 2) confirming that no deletions had occurred during the tagging event and the tagged plasmid had inserted at an EcoRI site.

Example 21

Characterization of Aspergillus oryzae Mutant HIN603 Rescued Locus pSMO603

The Aspergillus oryzae HIN603 rescued locus pSMO603 containing 1.0 kb was sequenced according to the procedure described in Example 2. The nucleic acid sequence (SEQ ID NO:39) as shown in FIG. 21 showed no homology to any published sequences.

A Southern blot of Aspergillus oryzae HowB430 genomic DNA digested with the restriction enzymes SphI, SalI, and BamHI was hybridized to probes made from both ends of the rescued plasmid. Probe 3 was generated by PCR using primer 970858 and primer 970859 shown below synthesized with an Applied Biosystems Model 394 DNA/RNA Synthesizer according to the manufacturer's instructions. Probe 4 was generated by PCR using primer 970860 and primer 970861. The template for the 50 μPCR labeling reaction was 10 ng of the rescued plasmid pSMOH603. PCR cycles and conditions were as described in Example 20. Southern conditions were as described in Example 20.

970858: 5'-TGTAGTCTGACTAGCATG-3' (SEQ ID NO:40)

970859: 5'-GGATCTTCACCTAGATCC-3' (SEQ ID NO:41)

970860: 5'-CATAGTGTCGACCAAGC-3' (SEQ ID NO:42)

970861: 5'-CAATCGAGCTTGCCTATG-3' (SEQ ID NO:43)

Different banding patterns on the Southern suggested that a deletion had taken place where the tagging occurred. When 500 ng of Aspergillus oryzae HowB430 genomic DNA was used as a template in a PCR reaction with primers 090858 and 090860, a 3 kb product was amplified suggesting a 2.5 kb deletion had occurred. Southern analysis of genomic DNA prepared from the tagged Aspergillus oryzae HIN603 strain, digested with HindIII, and probed with the NheI fragment from Aspergillus oryzae pyrG suggested that the tagged mutant had not been generated by a REMI event.

Example 22

Construction of Aspergillus oryzae HowB432

Aspergillus oryzae HowB432 was generated by transformation of Aspergillus oryzae JaL250 with a linear fragment containing the NA2-tpi promoter, a cellulase gene from Humicola lanuginosa (CAREZYME™ gene, Novo Nordisk A/S, Bagsv.ae butted.rd, Denmark), and the AMG terminator obtained from plasmid pGAG3 (FIG. 22).

Aspergillus oryzae JaL250 was constructed from Aspergillus oryzae JaL142 (Christensen et al., 1988, Bio/Technology 6: 1419-1422) by deleting the neutral protease I gene (npI). The npl deletion plasmid was constructed by exchanging a 1.1 kb BalI fragment coding for the central part of the npI gene in plasmid pJaL389 (FIG. 23), which contained a 5.5 kb SacI genomic fragment encoding the npI gene, with a 3.5 kb HindIII fragment from pJaL335 (FIG. 24) containing the pyrG gene flanked by repeats, thereby creating plasmid pJaL399 (FIG. 25). Aspergillus oryzae JaL142 was transformed with the 7.9 kb SacI fragment. Transformants were selected by relief of the uridine requirement on Minimal medium plates. The transformants were analyzed by Southern analysis as described in Example 14 and by IEF protease profile analysis according to standard methods.

Two out of 35 transformants possessed an altered Southern profile compared to the parent strain and displayed no neutral protease I activity by IEF. Furthermore, Southern analysis showed that one of the two transformants had a clean deletion of the npI gene and was designated Aspergillus oryzae JaL228.

Totally, 2.3×10⁷ conidiospores of Aspergillus oryzae JaL228 were spread on Minimal medium plates supplemented with 0.1% 5-fluoro-orotic acid (FOA) and 10 mM uridine. Eight FOA resistant colonies were obtained. A Southern blot of BamHI digested genomic DNA from the eight colonies probed with a 401 bp pyrG repeated region demonstrated that the pyrG gene had been excised by recombination at the repeated regions. Aspergillus oryzae JaL228 showed two bands of the expected size of 2.7 and 3.1 kb originating from the two copies of the repeated region. If the pyrG gene had been lost by recombination between the repeated regions, the 3.1 kb band would have disappeared and only the 2.7 kb would have remained. All 8 FOA resistant colonies showed this pattern of bands. Sequencing of a PCR fragment covering the junctions between the npI gene and the copy of the 401 bp repeat remaining in the 8 colonies confirmed that the pyrG gene was excised by recombination between the repeats. One of the colonies was designated Aspergillus oryzae JaL250.

pGAG3 was constructed by isolating from pDM176 (FIG. 26) a SwaI/PacI fragment containing the Humicola lanuginosa cellulase gene and ligating the fragment into SwaI/PacI digested pBANe6. The SwaI/PacI fragment from pDM176 and SwaI/PacI digested pBANe6 were separated on a 1% agarose gel, and isolated using a QIAquick Gel Extraction Kit (Qiagen Inc., Chatsworth, Calif.) according to the manufacturer's instructions prior to ligation. The ligation was used to transform E. coli DH5α cells, and the transformants were then screened by extracting plasmid DNA from the transformants using a QIAwell-8 Plasmid Kit according to the manufacturer's instructions, restriction digesting the plasmid DNA to confirm the presence of the correct size fragment, and sequencing the DNA according to the method described in Example 2.

pGAG3 was then digested with PmeI and the linear expression cassette was isolated by preparative agarose electrophoresis using TAE buffer. The linear cassette was then used to transform Aspergillus oryzae JaL250.

Transformation of Aspergillus oryzae JaL250 for amdS selection was conducted with protoplasts at a concentration of 2×10⁷ protoplasts per ml prepared as described in Example 2. Ten μg of the linear fragment described above were added to 100 μl of protoplasts. A volume of 250 μl of PEG (60% PEG 4000-10 mM CaCl₂ --10 mM Tris-HCl pH 8.0) was then added, and the mixture was placed at 37° C. for 30 minutes. Three ml of STC medium was added and the mixture was plated on Cove plates supplemented with 10 mM uridine for amdS selection. The plates were incubated 7-10 days at 34° C. Transformants were then transferred to plates of the same medium and incubated 3-5 days at 37° C. The transformants were purified by streaking spores and picking isolated colonies using the plates of the same medium without sucrose.

Example 23

Aspergillus oryzae Transformation with HpaI Linearized pDSY112 and Cellulase Expression Screening

Aspergillus oryzae HowB432 was transformed with HpaI digested pDSY112 and the transformants were recovered using the methods described in Example 5. Totally, 104 recovered transformants were grown in 24 well microtiter plates in 1/4 strength MY25. Samples were taken at 5 and 7 days and assayed for cellulase activity as described below.

Cellulase activity was measured according to the following protocol which is derived from Novo Nordisk method AF 302.1/1-GB which is available from Novo Nordisk A/S, Bagsv.ae butted.rd, Denmark upon request. A substrate solution containing 2% carboxymethylcellulose was prepared by dissolving the material in 100 mM MOPS pH 7.0 buffer at 80° C. for 10 minutes. CAREZYME™ (Novo Nordisk A/S, Bagsv.ae butted.rd, Denmark) was used as a standard. Stock solutions of 2.5 to 25 ECU per ml were prepared to construct a standard curve by diluting accordingly CAREZYME™ in 100 mM MOPS pH 7.0 buffer. Five μl aliquots of the standards and samples (diluted for shakeflasks and fermentations) were pipetted into individual wells of a 96 well plate. A volume of 65 μl of the 2% azocarboxymethylcellulose solution was pipetted into each of the wells and mixed. The reactions were incubated at 45° C. for 30 minutes and then stopped by the addition of 215 μl of stop reagent followed by mixing. The stop reagent was prepared by first suspending 0.2 g of ZnCl₂ in 20 ml of 250 mM MOPS pH 7.0 and adding the suspension to 80 ml of acidified ethanol containing 1.1 ml of concentrated HCI per liter of ethanol. The plate containing the stopped reaction was then centrifuged at 3000 rpm for 10 minutes. A 100 μl aliquot of each supernatant was pipetted into a 96 well plate and the absorbance measured at 600 nm. Using linear regression, the slope, intercept, and correlation coefficient were determined for the standards and samples.

The top ten cellulase producing transformants from the 24 well cultures were spore purified, and regrown in 24 well cultures as above and assayed for cellulase activity. The purified strains were also grown in MY25 in 125 ml shake flasks in MY25 pH 6.5 at 34° C. and samples were taken at 3 and 5 days for cellulase assays. Aspergillus oryzae HowB432 pDSY112 84-1-1 and Aspergillus oryzae HowB432 pDSY112 94-1-1 were also grown in fermentors (2 liters) as described in Example 8. Cellulase activities were measured as described above.

The results from the 24 well and shake flasks cultures are presented in Table 6 where the cellulase yield of Aspergillus oryzae HowB432 was normalized to 1.0.

                  TABLE 6     ______________________________________               24 well   Shake flasks                                    Fermentation results     Strain    (ECU/ml)  (ECU/ml)   (ECU/ml)     ______________________________________     HowB432   1.0       1.0        1.0     C112T50.1.1               2.0       0.9        NA     C112T84.1.1               2.1       2.0        1.3     C112T86.1.1               2.3       2.4        NA     C112T94.1.1               2.5       1.3        1.4     C112T95.1.1               2.1       1.3        NA     C112T100.1.1               2.3       1.6        NA     C112T101.1.1               1.9       1.6        NA     C112T102.1.1               2.0       2.1        NA     C112T103.1.1               1.5       2.0        NA     C112T104.1.1               2.2       2.3        NA     ______________________________________

A Southern blot of Aspergillus oryzae HowB432, Aspergillus oryzae DEBY599.3 and the PDSY112 transformants genomic DNAs digested with BglII was prepared and analyzed as described in Example 14 performed to determine whether pDSY112 had integrated at the homologous locus in the genome using the Aspergillus oryzae DEBY599.3 rescued flanking DNA as a probe.

A BGlII band of 2.7 kb from Aspergillus oryzae HowB432 hybridized with the probe, while an ˜8 kb BglII band from Aspergillus oryzae DEBY599.3 hybridized to the probe. In all of the transformants a wild-type BglII band of 2.7 kb and a second band corresponding to the transforming DNA hybridized to the probe. Therefore, none of the retransformants had exact gene replacements.

Example 24

Aspergillus oryzae Transformation with NdeI Linearized pDSY138 and Cellulase Expression Screening

Aspergillus oryzae HowB432 was transformed with NdeI digested pDSY138 using the method described in Example 5. Totally, 240 transformants were recovered which were grown in 24 well microtiter plates in 1/4 strength MY25 as described in Example 8 except samples were taken at days 3 and 5 and assayed for cellulase activity as described in Example 23. The top 20 cellulase producing transformants were spore purified and retested in 24 well microtiter cultures. The top 8 cellulase producing once purified transformants were spore purified a second time and tested in shake flasks in full-strength MY25 as described in Example 8. The top 2 producers were also grown in a 2 liter fermentor as described in Example 8. Cellulase activity was measured as described in Example 23.

The results obtained are shown in Table 7 below where the cellulase yield of Aspergillus oryzae HowB432 is normalized to 1.0.

                  TABLE 7     ______________________________________     Strain       ECU/ml  Southern Results     ______________________________________     HowB432      1.0     wild-type     C138T21.1.1  1.15    disrupted allele     C138T205.1.1 1.5     wild-type and disrupted alleles     ______________________________________

A Southern blot of Aspergillus oryzae DEBY932 Aspergillus oryzae HowB432, and pDSY138 genomic DNA preparations digested with NdeI was prepared and analyzed as described in Example 14 to determine if the pDSY138 DNA had integrated at the homologous locus producing gene replacements in the transformants using the Aspergillus oryzae DEBY932 rescued flanking DNA as a probe.

The results of the Southern blot demonstrated that an NdeI band of approximately 5 kb from Aspergillus oryzae HowB432 hybridized to the rescued locus while a band of approximately 10 kb from Aspergillus oryzae DEBY932 hybridized to the probe. In Table 7, the column labeled Southern results indicated whether the transformants had a wild-type NdeI fragment of the size observed in the parent strain Aspergillus oryzae HowB432 or whether the transformants had a band corresponding to the disrupted size observed in Aspergillus oryzae DEBY932.

Example 25

Aspergillus oryzae Transformation with AseI/PvuI palB Disruption Cassette from pMT1936 and Cellulase Screening

Aspergillus oryzae HowB432 was transformed using the same transformation procedure described in Example 5 with a 5.5 kb AseI/PvuI fragment containing the palB disruption cassette. The linear fragment for transformation was isolated by digestion of pMT1936 with AseI and PvuI and separation of the fragment on a 1% agarose gel using a QIAquick Gel Extraction Kit according to the manufacturer's instructions. The transformants obtained were then evaluated for growth on Minimal medium plates at pH 6.5 or pH 8.0.

The results showed that 10 of the 312 transformants tested were unable to grow at pH 8.0 indicative of the palB minus phenotype. The 10 palB minus transformants and 10 of the transformants that were able to grow at pH 8.0 were spore purified and tested in 24 well and shake flask cultures for cellulase production according to the procedures described in Example 23. The results tabulated in Table 8 below demonstrated that palB minus strains were better cellulase producers than the palB plus strains.

Southern blots of the genomic DNA from an Aspergillus oryzae palB minus mutant, an Aspergillus oryzae palB plus strain, and Aspergillus oryzae HowB432 were performed as described in Example 14 to determine if the AseI/PvuI transforming DNA fragment had integrated as a clean replacement into the palB locus.

The results of the Southern blot (Table 8) demonstrated that some of the palB minus strains had clean disruptions while others did not.

                  TABLE 8     ______________________________________                          Shake flasks     Strain   PalB phenotype                          (ECU/ml)   Southern pattern     ______________________________________     HowB432  plus        1.0        wild-type     CpalB5-1 plus        1.0        wild-type and other     CpalB6-1 plus        1.2        wild-type and other     CpalB7-1 plus        1.1        wild-type and other     CpalB24-1              plus        1.7        wild-type and other     CpalB28-1              minus       1.6        other     CpalB34-1              minus       1.6        disruption     CpalB45-1              minus       1.4        disruption     CpalB47-1              plus        1.1        wild-type and other     CpalB72-1              plus        1.3        wild-type and other     CpalB76-1              minus       1.6        disruption     CpalB89-1              minus       1.6        disruption     CpalB153-1              minus       1.1        disruption     CpalB161-1              plus        1.2        wild-type and other     CpalB163-1              minus       1.9        other     CpalB185-1              minus       1.8        other     CpalB190-1              minus       1.4        disruption     ______________________________________

Example 26

Aspergillus oryzae Transformation with AsnI/PvuI Manganese Superoxide Dismutase Disruption Cassette and Cellulase Screening

Aspergillus oryzae HowB432 was transformed with a 5.8 kb AsnI/Pvul fragment containing the manganese superoxide dismutase disruption cassette according to the same procedure described in Example 5.

Twenty transformants were obtained and tested for sensitivity to paraquat as described in Example 17. Seven of the 20 transformants were paraquat sensitive indicative of the manganese superoxide dismutase minus phenotype although they are sensitive to different levels of paraquat as indicated in Table 9 below. Those indicated as +++ for paraquat sensitivity are sensitive to as low as 2 mM paraquat, while those labeled - and ++ are not sensitive to paraquat and sensitive to intermediate levels of paraquat, respectively. All of the transformants were spore purified and tested in 24 well cultures for cellulase production as described in Example 23. The strains were also tested in 125 ml shake flasks cultures as described in Example 23. The results are shown in Table 9 below. The strains that are paraquat sensitive produce on average more CAREZYME™ than those strains that are not paraquat sensitive.

Southern blots of the transformants and Aspergillus oryzae HowB432 were prepared and analyzed as described in Example 14 to determine if the AsnI/PvuI manganese superoxide dismutase disruption cassette had integrated to give a clean replacement into the manganese superoxide dismutase disruption cassette locus.

The results of the Southern blot shown in Table 9 below indicate that the strains sensitive to 2 mM paraquat have only the disrupted locus of manganese superoxide dismutase, while those sensitive to intermediate levels of paraquat have both the wild-type locus and the disrupted cassette. The intermediate sensitivity to paraquat may be explained by the fact that manganese superoxide dismutase is a homodimer, and those cells expressing the wild-type and truncated manganese superoxide dismutase coded for by the disruption cassette would be producing heterodimers that are probably not functional.

                  TABLE 9     ______________________________________                          Shake flasks     Strain  Paraquat sensitivity                          (Relative LU/ml)                                      Southern Results     ______________________________________     HowB432 -            1.00        wild-type     432162T3             +++          1.26        disrupted     432162T7             ++           1.24        wild-type & other     432162T8             -            1.21        wild-type & other     432162T9             -            0.61        wild-type & other     432162T10             ++           1.14        wild-type & other     432162T11             ++           1.04        wild-type & other     432162T12             ++           1.12        wild-type & other     432162T15             -            0.44        wild-type & other     432162T16             ++           1.10        wild-type & other     432162T17             +++          1.53        --     432162T18             -            1.12        --     432162T19             +++          1.44        --     ______________________________________

Example 27

Construction of Glucose Transporter Gene Overexpression Plasmids pHB218 and pDSY153 and Stop Control Plasmids pDSY152 and pDSY155

Plasmids to overexpress the glucose transporter rescued locus from Aspergillus oryzae DEBY599.3 were constructed to determine if overexpression of the glucose transporter would lead to an increase in the yields of Humicola lanuginosa lipase and cellulase. The glucose transporter open reading frame was PCR amplified to place SwaI and PacI sites at the 5' and 3' end of the ORF, respectively. The following primers synthesized with an Applied Biosystems Model 394 DNA/RNA Synthesizer according to the manufacturer's instructions were used in combination with 0.2 μg of pDSY112 in the amplification:

961176: 5'-ATTTAAATGGTCCTCGGTGGATCAAGC-3' (SEQ ID NO:44)

961177: 5'-TTAATTAATTAGTCCTGTCTGCGCTGGT-3' (SEQ ID NO:45)

The conditions and parameters used for the amplification are described in Example 2. Ten μl of the PCR reaction was electrophoresed on an agarose gel, and a 1.5 kb product was obtained as expected. The PCR product was cloned using a pPCR-Script™ Kit (Stratagene, La Jolla, Calif.) according to the manufacturer's protocols. The ligation reaction was used to transform E. coli DH5α cells, and plasmid DNA was isolated from several of the transformants using the QIAWell-8 Plasmid Kit. The plasmids were digested with NotI and EcoRI to determine which clones had the 1.5 kb insert. Six of the 11 clones analyzed had the correct size insert as determined by electrophoreses on an agarose gel. One of the clones, pDSY119, was digested with PacI and SwaI, and the digest was run on an agarose gel. The 1.5 kb SwaI/Pac I band was excised from the gel, and DNA was purified from the gel slice using the QIAQuick Gel Extraction Kit. The 1.5 kb fragment was ligated with SwaI/PacI cut pBANe13 (FIG. 3) using standard conditions (Sambrook et al., 1989, supra). The ligation was used to transform E. coli DH5α cells, and plasmid DNA was isolated from several of the transformants. The plasmids were digested with SwaI/PacI to determine which clones had the expected 1.5 kb insert. The final plasmid was designated pHB218 (FIG. 27).

As a control for the overexpression experiments, a derivative of pHB218 in which a stop codon was inserted at amino acid 9 in the glucose transporter open reading frame was made using site-directed mutagenesis. A MORPH™ Site-Specific Plasmid DNA Mutagenesis kit from 5 Prime→3 Prime was used for the mutagenesis, and protocols provided with the kit were followed. The reaction contained pHB218 as template, and the mutagenic primer used was:

970545: 5'-CGGTGGATCAAGCGGTTAATTAATCACTCCGTACCTGAT-3' (SEQ ID NO:46)

Several E. coli colonies were obtained after following the protocols, and plasmid DNA was isolated from the colonies using the QIA-Well8 plasmid kit. The plasmids were digested with PacI since the mutagenic primer introduced a PacI site which served as a marker for the mutagenesis. Two of the plasmids with the extra PacI site indicative of a successful mutagenesis were sequenced as described in Example 2 to confirm the presence of the stop codon at amino acid 9 in the ORF. The pHB218 derivative with the stop codon at amino acid 9 was designated pDSY152.

Versions of pHB218 and pDSY152 in which the selectable marker was the bar gene were constructed for transformation of strains which are pyrG plus. The SwaI/PacI inserts from pHB218 and pDSY152 were isolated by restriction digestion, electrophoresed on an agarose gel, and purified using QIAQuick Gel Extraction Kit. The inserts were ligated into pSE39 (FIG. 28) and digested with SwaI/PacI. The ligation reaction was used to transform E. coli DH5α, and plasmid DNA was isolated from the colonies as described above. The plasmids were digested with SwaI/PacI to determine which clones contained the expected 1.5 kb insert. The plasmids were sequenced as described in Example 2 to confirm the presence or absence of the stop codon at amino acid 9 in pDSY155 and pDSY153 (FIG. 29), respectively. The only difference between pDSY155 and pDSY153 was the stop codon at amino acid 9 of the glucose transporter ORF in pDSY155.

Example 28

Transformation of Aspergillus oryzae HowB430 and Aspergillus oryzae HowB432 with pHB218 and pDSY152 and Lipase and Cellulase Screening, Respectively

Aspergillus oryzae HowB430 was transformed with pHB218 or pDSY152, and the transformants were recovered using the methods described in Example 5. One hundred and twenty transformants each with pHB218 and pDSY152 were recovered, grown in 24-well microtiter plates in 1/100 strength MY25 and assayed for lipase activity after 3 and 5 days as described in Example 8. The assay results showed that there was a slight shift towards higher lipase production in the pHB218 transformants versus the pDSY152 transformants supporting the idea that overexpression of the glucose transporter has a positive effect on lipase expression.

Aspergillus oryzae HowB432 was transformed with pHB218 and pDSY152, and the transformants were recovered using the methods described in Example 5. One hundred transformants each with pHB218 and pDSY152 were recovered, grown in 24-well microtiter plates in 1/4 strength MY25 and assayed for cellulase activity after 3 and 5 days as described in Example 23. The assay results showed that there was a shift towards higher cellulase production in the pHB218 transformants versus the pDSY152 transformants indicating that overexpression of the glucose transporter had a positive effect on cellulase expression.

Example 29

Transformation of Aspergillus oryzae DEBY10.3 with pDSY153 and pDSY155 and Lipase Screening

Aspergillus oryzae DEBY10.3 was transformed with pDSY153 and pDSY155, and the transformants were recovered using the methods described in Example 5. Two hundred sixteen and 144 transformants with pDSY153 and pDSY155, respectively, were recovered, grown in 24-well microtiter plates in 1/100 strength MY25, and assayed for lipase activity on days 4 and 6 as described in Example 8. There was shift towards higher lipase production in the pDSY153 transformants when compared to the pDSY155 transformants indicating that overexpression of the glucose transporter led to an increase in lipase production and also suggesting that the palB minus effect and the glucose transporter overexpression were additive.

Example 30

Identification of Tagged Event in Aspergillus oryzae HowL795

Genomic DNA was prepared from Aspergillus oryzae HowL795 according to Example 9. One μg of DNA was digested with either SnaB1 or NsiI. Both enzymes cleave within the pyrG gene contained on the tagging construct. The DNA was then diluted to 4 ng/μl and recircularized with T4 Ligase at 22° C. for 18 hours. Inverse PCR was then performed using approximately 500 ng of recircularized DNA using the primers shown below which were synthesized with an Applied Biosystems Model 394 DNA/RNA Synthesizer according to the manufacturer's instructions. Both were located downstream of the NsiI and SnaBI sites.

Primer x: 5'-GCACTCGAATGACTACT-3' (SEQ ID NO:47)

Primer y: 5'-CGCATCATACTTGCGACA-3' (SEQ ID NO:48)

The inverse PCR amplification reaction contained the following components: 500 ng of recircularized DNA, 150 pmoles of primer x, 150 pmoles of primer y, 1 mM each of dATP, dCTP, dGTP, and dTTP, 1×Taq polymerase buffer, and 2.5 U of Taq polymerase. The reaction was incubated in an Ericomp Thermal Cycler programmed as follows: One cycle at 95° C. for 5 minutes followed by 30 cycles each at 95° C. for 1 minute, 55° C. for 1 minute and 72° C. for 2 minutes. The PCR product was isolated by electrophoresis on a 1% agarose gel.

PCR of the religated SnaBI DNA amplified a 4 kb fragment whereas PCR of the religated NsiI DNA amplified a 2 kb fragment. PCR confirmed that the smaller NsiI fragment was contained within the larger SnaBI fragment.

DNA sequence analysis was performed according to the procedure described in Example 2 using primer A. The analysis identified that the insertion of pSO122 had occurred in the 3' non-translated region of the amdS gene contained within plasmid pBANe8.

Example 31

Construction of Aspergillus oryzae MStr107

Aspergillus oryzae MStr107 was constructed to contain extra copies of one of the native alpha-amylase (TAKA) genes (FUNGAMYL™ gene, Novo Nordisk A/S, Bags.ae butted.rd, Denmark), by transforming Aspergillus oryzae HowB101 with a DNA fragment from pMStr15. pMStr15 was constructed from pCaHj505 and pTAKA17 as described below. Standard methods were employed (Sambrook et al., 1989, supra) except where noted.

pCaHj505 (FIG. 30) was constructed to contain the Aspergillus oryzae NA-14 alpha-amylase (TAKA) promoter, the Aspergillus niger glucoamylase (AMG) terminator, and the Aspergillus nidulans amdS gene from the following fragments:

a) The vector pToC65 (WO 91/17243) digested with EcoRI and XbaI.

b) A 2.7 kb XbaI fragment from Aspergillus nidulans carrying the amdS gene (Corrick et al., 1987, Gene 53: 63-71). The amdS gene was used as a selective marker in fungal transformations. The amdS gene was modified so that the BamHI site normally present in the gene was destroyed. This was done by introducing a silent point mutation using the primer: AGAAATCGGGTATCCTTTCAG (SEQ ID NO:49).

c) A 0.6 kb EcoRI-BamHI fragment carrying the Aspergillus oryzae NA-14 alpha-amylase promoter.

d) A 675 bp XbaI fragment carrying the Aspergillus niger glucoamylase transcription terminator. The fragment was isolated from the plasmid pICAMG/Term (EP 238 023).

The BamHI site of fragment c was connected to the XbaI site in front of the transcription terminator on fragment d via the pIC19R linker (BamHI to XbaI) (Boehringer Mannheim, Indianapolis, Ind.).

pMStr15 (FIG. 31) was constructed to contain the Aspergillus oryzae NA-14 alpha-amylase promoter, gene and terminator and the Aspergillus nidulans amdS gene. The alpha-amylase gene with promoter and terminator was excised from pTAKA17 (European patent 0238 023) as a 2.9 kb EcoRI-HindIII fragment and cloned adjacent to the amdS gene in the vector pCaHj505 by replacing the EcoRI-XbaI promoter/terminator fragment in pCaHj505. To facilitate cloning, the recessed 3' termini generated by HindIII and XbaI digestion were filled in.

A single linear DNA fragment containing both the alpha-amylase gene and the amdS gene was obtained by digesting pMStrl5 with NotI, resolving the vector and insert sequences using agarose gel electrophoresis, excising the appropriate DNA band from the gel, and purifying the DNA from the agarose using GenElute™ Agarose Spin Columns according to the manufacturer's directions (Supelco, Bellefonte, Pa.). This 5.6 kb NotI fragment was used to transform Aspergillus oryzae HowB101 to construct Aspergillus oryzae MStr107, using the transformation protocol and selective medium described in Example 2. Transformants were propagated from single colonies twice in succession on COVE medium with 0.1% Triton X100 before performing additional screens.

Aspergillus oryzae MStr107 was selected from among the transformants based on its ability to produce more alpha-amylase than Aspergillus oryzae HowB101. The ability of the transformants to produce alpha-amylase was determined by culturing them in 10 ml of YPM medium for 4 days at 30° C. with shaking and resolving 5 μl of the culture medium by SDS-PAGE according to standard methods. The strain producing the most alpha-amylase under these conditions was selected as Aspergillus oryzae MStr107, and was compared in a 3 liter fermentation culture to Aspergillus oryzae HowB101. The medium was composed of maltose syrup, yeast extract, KH₂ PO₄, K₂ SO₄, (NH₄)₂ SO₄, citric acid, MgSO₄, trace metals and uridine. Under these conditions, Aspergillus oryzae MStr107 produced 360% of Aspergillus oryzae HowB101.

Example 32

Aspergillus oryzae Mstr107 Transformation with Linearized pDSY82

Protoplasts of Aspergillus oryzae Mstr107 were prepared as described in Example 2. A 5-15 μl aliquot of pDSY82 (6 μg) linearized with 1.25 U of XbaI was added to 0.1 ml of the protoplasts at a concentration of 2×10⁷ protoplasts per ml in a 14 ml Falcon polypropylene tube followed by 250 μl of 60% PEG 4000-10 mM CaCl₂ --10 mM Tris-HCl pH 7, gently mixed, and incubated at 37° C. for 30 minutes. Three ml of SPTC were then added and the suspension was gently mixed. The suspension was mixed with 12 ml of molten overlay agar (1×COVE salts, 1% NZ amine, 0.8M sucrose, 0.6% Noble agar) or 3 ml of STC medium and the suspension was poured onto a Minimal medium plate. The plates were incubated at 37° C. for 3-5 days.

The transformation frequency of Aspergillus oryzae MStr107 with pDSY82 and XbaI was approximately 200 transformants/μg of DNA. A library of approximately 30,000 transformants was obtained. Spores from 70 pools with approximately 400 transformants in each pool were collected and stored in a 20% glycerol, 0.1% Tween 80 at -80° C. The pools and transformants from these libraries were designated with the letter "x".

Example 33

Characterization of Integration Events in "REMI" Aspergillus oryzae MStr107 Transformants

Transformants of Aspergillus oryzae MStr107 with pDSY82 and XbaI (library "x") were analyzed as described in Example 6. Genomic DNA was isolated from 40 transformants, 20 from one pool (x15) and 20 from 20 various pools. DNA samples were cut with HindIII, resolved, blotted and probed with radiolabeled pDSY82. Thirty-three of 40 displayed apparently novel band patterns, suggesting that plasmid integrations were distributed to different sites in the genome. For 19 of the 40 transformants the band patterns suggested that only one copy of pDSY82 integrated in the genome, while more than one copy was observed in the remaining 21 transformants. DNA from the 20 transformants taken from various pools was also cut with XbaI, resolved, blotted and probed with radiolabeled pDSY82 as described in Example 6. A single, plasmid-sized band was observed indicating REMI had occurred at an XbaI site in 9 of the transformants.

Example 34

FUNGAMYL™ Expression Screening

The Aspergillus oryzae MStr107 tagged mutant library "x" pools described in Example 32 were assayed for FUNGAMYL™ expression.

For 96-well plate screens, MTBCDYU medium was used. For 24-well plate methods, 4xMTBCDYU medium was used.

Primary 96-well plate screens involved the dilution of spores from distinct pools into MTBCDYU so that one spore on average was inoculated per well when 100 μl of medium was dispensed into the wells. After inoculation, the 96-well plates were grown for 3-4 days at 34° C. under static conditions. Cultures were then assayed for FUNGAMYL™ activity as described below. Mutants of interest were isolated twice on YPG or Cove plates, and single colonies transferred to Cove agar slants. Spores from Cove slants were inoculated into 24-well plates containing 4xMTBCDYU with approximately 10³ spores per well and grown under static conditions for 4 days at 34° C. Cultures were then assayed for FUNGAMYL™ activity as described below.

The FUNGAMYL™ assay substrate (4-nitrophenyl-alpha-D-maltoheptasid-4,6-O-ethyliden, EPS) was prepared as a 1/2 strength solution relative to the instructions given by the manufacturer (Boehringer Mannheim, Indianapolis, Ind.). The substrate was prepared in HEPES pH 7.0 buffer. A FUNGAMYL™ standard (FUNGAMYL™, Novo Nordisk A/S, Bagsv.ae butted.rd, Denmark) was prepared to contain 2 FAU/ml in HEPES pH 7.0 buffer. The standard was stored at -20° C. until use. FUNGAMYL™ stock was diluted appropriately to obtain a standard series ranging from 0.02 to 0.2 FAU/ml just before use. Broth samples were diluted in HEPES buffer and 25 μl aliquots were dispensed to wells in 96-well plates followed by 180 μl of diluted substrate. Using a plate reader, the absorbance at 405 nm was recorded as the difference of two readings taken at approximately 1 minute intervals. FUNGAMYL™ units/ml (FAU/ml) were calculated relative to the FUNGAMYL™ standard solutions.

The results of the 96-well screen followed by the 24-well screen identified for further evaluation 51 transformants from the pDSY82 and XBaI transformations. These identified transformants produced higher levels of FUNGAMYL™ than the control strain Aspergillus oryzae MStr107.

Example 35

Shake Flask and Fermentation Evaluation

The highest FUNGAMYL™-producing DNA-tagged mutants described in Example 34 were evaluated in shake flasks and fermentors.

Shake flask evaluations were performed by inoculating half the spore content of a COVE slant suspended in a suitable volume of sterile water containing 0.02% TWEEN-80 into 100 ml of G1-gly medium at pH 7.0 in a 500 ml shake flask. The G1-gly shake flasks were incubated at 34° C. for 1 day at 270 rpm. Next, 5 ml of the G1-gly cultures were inoculated into 100 ml of 1/5MDU2BP at pH 6.5 in 500 ml shake flasks. Samples were taken at day 3, and FUNGAMYL™ activity was measured as described in Example 34.

The DNA-tagged mutant X70-25 and 257D11 were grown in a 3 liter lab fermentor containing a medium composed of Nutriose, yeast extract, MgSO₄, KH₂ PO₄, citric acid, K₂ SO₄, (NH₄)₂ SO₄ and trace metals solution. The fermentation was performed at a temperature of 34° C., a pH of 7, and the agitation was maintained between 1000-1200 rpm for 5 days. FUNGAMYL™ activity was measured by partial degradation of EPS to G₂ and G₃ derivatives (G=glucose). The G₂ and G₃ derivatives were then degraded to glucose and yellowish colored p-nitrophenolate anion by the addition of a surplus of alpha-glucosidase. The analytical output was determined as the change in absorbance at 405 nrm per unit time (3 minutes) at 37° C. and pH 7.1 after a preincubation for 2.5 minutes. FUNGAMYL™ was used as standard.

The results obtained are shown in Table 10 below where the FUNGAMYL™ yield of Aspergillus oryzae MStr107 as a control is normalized to 1.0.

                  TABLE 10     ______________________________________     FUNGAMYL ™ Expression by DNA Tagged Mutants                             24-well                                    Shake Flask                             in 96-well                                    Plate   Fermentation     Strain         #        Plates Results Results     Description             Pool   Screened (FAU/ml)                                    (FAU/ml)                                            (FAU/ml)     ______________________________________     HowB101 NA     NA       0.4    0.4     0.3     MStr107 NA     NA       1.0    1.0     1.0     X70-25  ×70                    580      1.4    1.3     1.2     257D11  ×6                    480      1.5    1.4     1.0     X70-42  ×70                    580      1.3    1.3     ND     263A3   ×6                    480      1.4    1.6     ND     X69-246 ×69                    350      1.6    1.4     ND     X59-122 ×59                    580      1.3    1.3     ND     X49-233 ×49                    460      1.4    1.4     ND     ______________________________________

As shown in Table 10, the mutants produced approximately 30-60% more FUNGAMYL™ than the control strain Aspergillus oryzae MStr107 when grown in 24-well plates and when grown in shake flasks. The mutant, Aspergillus oryzae X70-25 produced approximately 20% more FUNGAMYL™ than the control strain Aspergillus oryzae MStr107 when grown in fermentors.

Example 36

Screening for Morphological Mutants

The 5 "e" pools and 5 "b" pools described in Example 5 were screened for altered morphology by plating on CM-1 agar and incubating at 34° C. for 4 days.

Twenty-four colonies having altered plate morphology and covering the morphological variation within the pool were transferred to fresh CM-1 plates and incubated 5 days at 34° C. for single colony isolation. Each morphology (in most cases 1) on a plate, was transferred from a single colony to the center of a CM-1 plate and a PDA plate, and incubated 6-8 days at 34° C. before the morphology was evaluated, i.e., the diameter and the appearance. A total of 218 morphological mutants was transferred to COVE plates and incubated at 34° C. for 1-2 week to generate spores.

Example 37

Evaluation of Morphological Mutants

The morphological mutants isolated in Example 36 were evaluated in 24-well plates for lipase production according to the procedure described in Example 7. The highest yielding mutants were compared with respect to plate morphology on CM-1 agar, and 23 mutants covering the observed morphological variation were further tested in shake flasks containing CD medium to evaluate lipase production.

Approximately 0.25 ml of spore suspension from a CM-1 plate was inoculated into 25 ml of Gi-gly medium in a 125 ml PP flask and incubated at 34° C. for 24 hours. Then 0.5 ml of the 24 hour seed flask was transferred to 50 ml of CD medium supplemented with 1.0 μl of FUNGAMYL™ 800L (Novo Nordisk A/S, Bagv.ae butted.rd, Denmark) in a 125 ml PP flask, and incubated at 34° C., 200 rpm. The culture was sampled after 2 and 3 days and assayed for lipase activity as described in Example 7.

The isolated mutants were also tested in the following manner in oxygen limited media. Aspergillus oryzae HowL536.3 was run as a control since the strain possessed the wild type morphology and did not require uridine for growth. Approximately 250 μl of spore suspension was inoculated into a 125 ml shake flask containing 25 ml OL-1 medium and incubated at 34° C., 200 rpm until residual glucose was <<1 g/l measured using DIASTIX™ (Bayer, Elkhart, Ind.). Then 75 ml OL-6 medium was added to each flask and further incubated at 34° C., 200 rpm for approximately 25 hours until residual glucose in the Aspergillus oryzae HowL536.3 flask was approximately 5 g/l. At that time, all the flasks were assayed for residual glucose, and the flasks with significantly lower glucose (0-2 g/l) were considered positive. The majority of the flasks averaged around 5-10 g/l.

Twenty mutants converting the glucose faster than average were considered likely to be easier to aerate and were further tested in shake flasks containing CD medium as described above to evaluate lipase expression.

Based on these tests, 14 mutants were identified and further evaluated in lab fermentors according to the procedure described in Example 8. The morphological mutants listed below in Table 11 were identified.

                  TABLE 11     ______________________________________     Morphological mutants     Strain           Construction Pool   Description     ______________________________________     P2-7.1           pDSY82 + BamHI                        b2     colonial, easy to aerate, 50% yield                               increase in fermentors     P3-2.1           pDSY82 + BamHI                        b3     flat, yield 40% yield increase in                               fermentors     P4-8.1           pDSY82 + BamHI                        b5     easy to aerate, 30% yield increase                               in fermentors     P5-7.1           pDSY82 + BamHI                        b6     easy to aerate, 60% yield increase                               in fermentors     P7-14.1           pDSY81 + EcoRI                        e2     colonial, easy to aerate, 50% yield                               increase in fermentors     P8-10.1           pDSY81 + EcoRI                        e3     easy to aerate, yield increased 50%                               in fermentors     ______________________________________

Example 38

Rescue of Plasmid DNA and Flanking DNA from Morphological Mutants

The plasmid DNA and genomic flanking loci were isolated from mutants Aspergillus oryzae P4-8.1 and P7-14.1 using the procedure described in Example 9 except for the restriction endonuclease(s) used. Transformant E. coli HB11 p4-8.1 contained a BglII rescued locus from mutant Aspergillus oryzae P4-8.1. Transformant E. coli HB101 p7-14.1 contained a NarI rescued locus from mutant Aspergillus oryzae P7-14. 1.

The plasmid DNA and genomic flanking loci were isolated from mutants Aspergillus oryzae DEBY7-17.2, DEBY3-2.1, DEBY5-7.1, and DEBY8-10.1. The rescued plasmids were generated as previously described in Example 9 with the exception that rescues pSMO717, pSMO321, pHowB571, and pSMO810 were isolated from transformed E. coli DH5α cells.

Transformant E. coli DH5α pSMO717 contained the BglIl rescued locus from mutant Aspergillus oryzae DEBY7-17.2. Transformant E. coli DH5a pSMO321 contained the BglII rescued locus from mutant Aspergilus oryzae DEBY3-2.1. Transformant E. coli DH5α pHowB571 contained the NdeI rescued locus from mutant Aspergillus oryzae DEBY5-7.1. Transformant E. coli DH5α pSMO810 contained the NdeI rescued locus from mutant Aspergillus oryzae DEBY8- 10.1.

Example 39

Characterization of Morphological Mutant Aspergillus oryzae P4-8.1 Rescued Locus p4-8.1

The Aspergillus oryzae P4-8.1 rescued locus p4-8.1 containing 915 and 665 bp regions on either side of the integration event was sequenced according to the procedure described in Example 2. The nucleic acid sequence (SEQ ID NO:50) and the deduced amino acid sequence (SEQ ID NO:51) are shown in FIG. 32. The nucleic acid sequence suggested that the integration event occurred within an open reading frame for a homologue of the Saccharomyces cerevisiae YHM4 Heat Shock protein gene. The deduced amino acid sequence (SEQ ID NO:51) showed 40.2% identity to the Saccharomyces cerevisiae YHM4 Heat Shock protein (SEQ ID NO:52) and 41.8% identity to a Schizzosaccharomyces pompe Heat Shock Protein 70 (SEQ ID NO:53).

Example 40

Aspergillus oryzae Transformation with BglII Linearized p4-8.1 and Morphology Screening

To verify the link between the observed plate morphology for Aspergillus oryzae P4-8.1 and the rescued genomic locus, Aspergillus oryzae HowB430 was transformed with the BgllI linearized rescued locus of Aspergillus oryzae P4-8.1, p4-8.1, using the procedure described in Example 5.

Sixty-six transformants were obtained, transferred to CM-1 agar, and incubated at 34° C. for 3-4 days to evaluate the morphology. Sixteen transformants with the correct plate morphology were transferred to fresh CM-1 plates as center colonies, and 12 transformants maintaining the plate morphology after 4 days at 34° C. were analyzed by Southern blot analysis with a PCR amplified 300 bp fragment of the rescued locus as a probe. The fragment was PCR amplified using the primers below synthesized with an Applied Biosystems Model 394 DNA/RNA Synthesizer according to the manufacturer's instructions.

HSP-1: 5'-TACGGTTGACAGTGGAGC-3' (SEQ ID NO:54)

HSP-3r: 5'-CACTGACTTCTCCGATGC-3' (SEQ ID NO:55)

The amplification reaction (50 μl) contained the following components: 0.2 ng of p4-8.1, 50 pmol of primer HSP-1, 50 pmol of primer HSP-3r, 0.25 mM each of dATP, dCTP, dGTP, and dTTP, 1×Taq polymerase buffer, and 2.5 U of Taq polymerase. The reaction was incubated in an Ericomp Thermal Cycler programmed as follows: One cycle at 95° C. for 3 minutes; 30 cycles each at 95° C. for 1 minute, 58° C. for 1 minute, and 72° C. for 1.5 minutes; and 1 cycle at 72° C. for 5 minutes. The PCR product was isolated by electrophoresis on a 1% agarose gel.

Samples of the genomic DNA were obtained from each of the 12 transformants obtained according to the method described in Example 9. The genomic DNAs were digested with BglII and submitted to Southern analysis according to the procedure described in Example 14.

All 12 transformants were affected at the rescued locus, suggesting a connection between this locus and the observe d plate morphology.

Example 41

Characterization of Morphological Mutant Aspergillus oryzae P7-14.1 Rescued Locus P7-4.1

The Aspergillus oryzae P7-14.1 rescued locus p7-14.1 containing 1040 and 520 bp regions on either side of the integration event was sequenced according to the procedure described in Example 2. The nucleic acid sequence (SEQ ID NO:56) and the deduced amino acid sequence (SEQ ID NO:57) are shown in FIG. 33. The nucleic acid sequence suggested that the integration event occurred within an open reading frame for a homologue of the Aspergillus nidulans chitin synthase B (chsB) gene and the Aspergillus fumigatus chitin synthase G (chsg) gene. Identities of 94% and 80% were found when the deduced amino acid sequences of the two sides of the rescued locus (SEQ ID NO:57), the chsB gene (SEQ ID NO:58), and the chsG gene (SEQ ID NO:59) were compared.

Disruption of the chsB gene in Aspergillus nidulans is known to change the morphology significantly (Yanai et al., 1994, Biosci. Biotech. Biochem. 58: 1828-1835), and in Aspergillus fumigatus disruption of the chsG gene is known to cause colonial morphology (Mellado et al., 1996, Molecular Microbiology 20: 667-679), which is the observed phenotype of Aspergillus oryzae P7-4.1.

Example 42

Aspergillus oryzae Transformation with a Linear chs Fragment and Morphology Screening

A 1.9 kb DNA fragment was generated by PCR using as the template Aspergillus oryzae HowB430 genomic DNA prepared as described in Example 6. Primer A, 5' CACCAAGTCAGAGCGTC-3' (SEQ ID NO:60), was derived from the rescued chs Aspergillus oryzae homology. Primer 5, 5'-GGICCITTYGAYGAYCCICA-3' (SEQ ID NO:61), was degenerate based on the consensus sequence of the Aspergillus fumigatus chsG and Aspergillus nidulans chsB genes. The amplification reaction (50 μl) contained the following components: 10 ng of pHB220, 48.4 pmol of each primer, 1 mM each of dATP, dCTP, dGTP, and dTTP, and the Advantage-GC™Tth Polymerase Mix (Clontech, Palo Alto, Calif.). The reaction was incubated in an Ericomp Thermal Cycler programmed as follows: One cycle at 95° C. for 3 minutes; 30 cycles each at 95° C. for 1 minute, 58° C. for 1 minute, and 72° C. for 3 minutes; and 1 cycle at 72° C. for 5 minutes. The PCR product was isolated by electrophoresis on a 1% agarose gel.

The DNA fragment was cloned into the PCR-Blunt Cloning Vector (Invitrogen, San Diego, Calif.). A HindIII site in the multicloning site was destroyed by filling in with the Klenow fragment of DNA Polymerase I. A 2 kb HindIII-EcoRI fragment containing the Basta gene conferring resistance to Bialaphos was obtained from pMT1612 (FIG. 34) and inserted into the chs HindIII site located approximately 0.7 kb within the chs fragment. The resultant plasmid was labelled pHB220.

Using pHB220 as template, a 4 kb PCR fragment was generated using primer A and 5'-GGGCCGTTTGACAATCCGCAT-3' (SEQ ID NO:62). The amplification reaction was performed as described above except the reaction was incubated in an Ericomp Thermal Cycler programmed as follows: One cycle at 95° C. for 3 minutes; 30 cycles each at 95° C. for 1 minute, 58° C. for 1 minute, and 72° C. for 6 minutes; and 1 cycle at 72° C. for 5 minutes. The PCR product was isolated by electrophoresis on a 1% agarose gel.

The PCR fragment was then used directly to transform protoplasts prepared from Aspergillus oryzae HowL795 according to the procedure described in Example 5. Of 100 transformants, three transformants appeared to have a "colonial" plate morphology on Minimal medium plates and PDA plates.

Southern analysis was performed on the three transformants and Aspergillus oryzae HowB430 as a control using the 2 kb DIG-labelled chs fragment as probe. Genomic DNA was prepared from the three strains and control strain as described in Example 9. Samples of the genomic DNA from each of the 3 transformants digested with HindIII was submitted to Southern analysis according to the procedure described in Example 14.

The Southern analysis showed that each of the three transformants had undergone a gene replacement substituting the chs/basta construct with the wild-type chs gene. The results confirmed that the colonial morphology observed in the chs tagged strain Aspergillus oryzae P7-14.1 was associated with a mutation of the chs gene.

An apparent effect of the chs gene on colony morphology was observed in shake flask cultures containing MY25 medium performed as described in Example 8. The pellet mass of the colonies in the broth appeared less dense in the chs mutants of Aspergillus oryzae HowL795 compared to Aspergillus oryzae HowL795.

Fermentations were also performed as described in Example 8 on two derivatives of Aspergillus oryzae HowL795 containing gene disruptions of the chs gene. Lipase yields in both strains were approximately 21% greater than Aspergillus oryzae HowL795. The kinetics of enzyme production appeared to be increased in the chs mutants in the later stages of fermentation suggesting that these strains exhibited a more optimal tank morphology.

Example 43

Characterization of Morphological Mutant Aspergillus oryzae DEBY7-17.2 Rescued Locus pSMO717

The Aspergillus oryzae DEBY7-17.2 rescued locus pSMO717 containing 400 bp was sequenced according to the method described in Example 2. The nucleic acid sequence (SEQ ID NO:63) and the deduced amino acid sequence (SEQ ID NO:64) are shown in FIG. 35. The deduced amino acid sequence (SEQ ID NO:64) showed 44% identity to the deduced amino acid sequence of an ORF of Aspergillus nidulans (AC000133) (SEQ ID NO:65).

Example 44

Characterization of Morphological Mutant Aspergillus oryzae DEBY3-2.1 Rescued Locus pSMO321

The Aspergillus oryzae DEBY3-2.1 rescued locus pSMO321 containing 1.0 kb was sequenced according to the method described in Example 2. The nucleic acid sequence (SEQ ID NO:66) shown in FIG. 36 showed no homology to any published sequences.

Probes from either end of the rescued plasmid pSMO321 were generated by PCR. Probe 5 was generated with primers 970850 and 970851 shown below. Probe 6 was generated with primers 970852 and primer 970853 shown below. The primers were synthesized with an Applied Biosystems Model 394 DNA/RNA Synthesizer according to the manufacturer's instructions. The template for the 50 μl PCR labeling reaction was 10 ng of the rescued plasmid pSMO321. PCR cycles and conditions were as described in Example

970850: 5'-GTTCTATTGAGATACGCG-3' (SEQ ID NO:67)

970851: 5'-ACAAGCCGACCGGTTTTG-3' (SEQ ID NO:68)

970852: 5'-CGATAAGGACTCCAAGAG-3' (SEQ ID NO:69)

970853: 5'-GTCGCGCATAATATGAAG-3' (SEQ ID NO:70)

Southern blots of Aspergillus oryzae HowB430 genomic DNA digested with SphI, SalI and BamHI were prepared and analyzed according to the method described in Example 14. The blots were hybridized independently to probes 5 and 6 made from the ends of the rescued plasmid.

Analysis of the Southern blots suggested no deletions had occurred. When PCR was performed using 500 ng of genomic DNA from Aspergillus oryzae HowB430 with primers 090850 and 090852, a 500 bp product was amplified as predicted verifying that no deletions had taken place.

Genomic DNA was prepared from the tagged mutant strain Aspergillus oryzae DEBY3-2.1 as described in Example 9, digested with the restriction enzyme used for REMI (BamHI), blotted and probed with the NheI fragment from Aspergillus oryzae pyrG. Southern analysis of this blot suggested the tagged plasmid had inserted into a BamHI site in the genome.

Example 45

Characterization of Morphological Mutant Aspergillus oryzae DEBY 5-7.1 Rescued Locus pHowB571

The Aspergillus oryzae DEBY5-7.1 rescued locus pHowB571 containing 600 bp was sequenced according to the method described in Example 2. The nucleic acid sequence (SEQ ID NO:71) shown in FIG. 37 showed no homology to any published sequences.

To test if a deletion occurred during tagging, a Southern blot was prepared and analyzed according to the method described in Example 14 using genomic DNA from Aspergillus oryzae HowB430 digested with SphI, SalI and BamHI. Probes from either end of the rescued tagged plasmid pSMO571 were generated by PCR. Probe 7 was generated with primer 970936 and primer 970937 shown below. Probe 8 was generated with primer 970938 and primer 970939 shown below. The primers were synthesized with an Applied Biosystems Model 394 DNA/RNA Synthesizer according to the manufacturer's instructions. The template for the PCR labeling reaction was 10 ng of pSMO571. PCR cycles and conditions were as described in Example 20.

970936: 5'-CTTCCTCATAAACCACCC-3' (SEQ ID NO:72)

970937: 5'-AACTGACAGGACAAGACC-3' (SEQ ID NO:73)

970938: 5'-GACTTGCATCACTTCCTC-3' (SEQ ID NO:74)

970939: 5'-TGAAGCTGAGAGTAGGTG-3' (SEQ ID NO:75)

The results showed identical banding patterns from both probes suggesting no deletions had occurred. PCR was used to verify that no deletions had occurred using the method described in Example 20. A total of 500 ng of genomic DNA from Aspergillus oryzae HowB430 was used as template with primer 970936 and primer 970939. A 550 bp product was amplified as predicted. Southern data from genomic DNA obtained from the tagged mutant Aspergillus oryzae DEBY5-7.1 digested with BamHI, hybridized to the NheI fragment of pyrG suggested that the tagged plasmid had inserted into a BamHI site in the genome. Southern blot conditions are described as above.

Southern and PCR analysis demonstrated the tagged plasmid had inserted directly into a BamHI site. Cloning with TOPO pCRII vector and subsequent sequencing according to Example 2 of the PCR product generated using Aspergillus oryzae HowB430 genomic DNA with primers from the rescued ends of Aspergillus oryzae DEBY5-7.1 confirmed this result.

Example 46

Characterization of Morphological Mutant Aspergillus oryzae DEBY8-10.1 pSMO810

The Aspergillus oryzae DEBY8-10.1 rescued locus pSMO810 containing 750 bp was sequenced according to the method described in Example 2. The nucleic acid sequence (SEQ ID NO:76) shown in FIG. 38 showed no homology to any published sequences.

Probes from either end of the rescued plasmid pSMO810 were generated by PCR. Probe 9 was generated with primer 970854 and primer 970855 shown below. Probe 10 was generated with primer 970856 and primer 970857 shown below. The primers were synthesized with an Applied Biosystems Model 394 DNA/RNA Synthesizer according to the manufacturer's instructions. The template for the PCR labeling reaction was 10 ng of pSMO810. The PCR reaction and conditions were as described in Example 20.

970854: 5'-GTTTCGGTATTGTCACTG-3' (SEQ ID NO:77)

970855: 5'-ACAGGTGAACAACTGAGG-3' (SEQ ID NO:78)

970856: 5'-CGACCAAACTAGACAAGC-3' (SEQ ID NO:79)

970857: 5'-CTTTCCTCTTGGACACAC-3' (SEQ ID NO:80)

Southern analysis was performed as described in Example 14. Southern analysis of genomic DNA from Aspergillus oryzae HowB430 digested with SphI, SalI, and BamHI hybridized independently with probes 9 and 10 suggested no deletions had occurred during the insertion of the tagged plasmid. PCR using 500 ng of Aspergillus oryzae HowB430 genomic DNA as template with primers 090854 and 090856 amplified a 500 bp product as expected with no deletions. Southern analysis of genomic DNA prepared from mutant Aspergillus oryzae DEBY8-10.1, digested with EcoRI, and probed to the NheI fragment of pyrG suggested that the plasmid integrated into an EcoRI site in the genome. Cloning with TOPO pCRll vector and subsequent sequencing according to Example 2 of the PCR product generated using Aspergillus oryzae HowB430 genomic DNA with primers from the rescued ends of Aspergillus oryzae DEBY8-10.1 confirmed this result.

Example 47

Screening on a Poor Carbon Source for High Producers

Eighteen pools of Aspergillus oryzae HowB430 transformants, 11 generated with HindIII digested pDSY81 and in the presence of HindIII, and 7 generated with SalI digested pDSY81 and in the presence of SalI (see Example 5) were screened on poor carbon sources to identify mutants which were high producers of lipase. Glycerol was used as poor carbon source, since the expression of lipase is very low on glycerol, but a number of other carbon sources, e.g., xylose, sucrose, and polyols such as mannitol and sorbitol could be used in a similar way.

The primary 96-well plate screen was performed as described in Example 7, but with GLY25 medium composed of 100 ml of 10% yeast extract, 100 ml of 25% glycerol, 100 ml of 2% urea per liter diluted 50-fold. Lipase assays were performed as described in Example 7.

Mutants of interest were then inoculated directly into 24-well plates containing the same medium as above and grown 6 days at 34° C. and 100 rpm. Cultures were then assayed for lipase activity as described in Example 7, and mutants of interest were plated on COVE plates to produce spores, spread on PDA plates to produce single colonies, and then 4 single isolates of each mutant were grown on CM-glycerol agar (as CM-1 agar, but maltose was replaced by glycerol as carbon source) to produce spores for inoculation of 24-well plates as above.

After the 24-well plates, 10 transformants were identified for further evaluation in shake flasks. The shake flasks contained 50 ml of medium at pH 6.5 composed of 1 g of MgSO₄ --7H₂ O, 1 g/l K₂ SO₄, 15 g of KH₂ PO₄, 0.25 ml of trace metals solution, 0.7 g of yeast extract, 3 ml of 50% urea, 2 ml of 15% CaCl₂ --2H₂ O, and 2% carbon source (either maltose, glucose, sucrose, or glycerol). The shake flasks containing 50 ml of medium in a 125 PP flask were inoculated with 0.5 ml G1-gly overnight culture, incubated at 34° C. and 200 rpm, and sampled after 2 and 3 days. Lipase activity was measured as above.

The results are shown in Table 12 where lipase production by Aspergillus oryzae HowB430 grown on glycerol as carbon source is normalized to 1.0.

                  TABLE 12     ______________________________________     Strain   Pool    Glycerol  Maltose                                       Glucose                                             Sucrose     ______________________________________     HowB430  NA      1.0       152    44    1.1     HINL880.1              Hin-5   1.5       182    49    10     HINL895.3              Hin-20  8.4       140    38    11     HINL918.4              Hin-24  5.7       163    45    8.8     SALL587.4              Sal-4   1.6       178    89    22     SALL591.2              Sal-4   1.3       232    126   33     SALL631.2              Sal-5   16.6       72    35    25     SALL631.3              Sal-5   18.3       67    42    29     SALL664.2              Sal-15  6.1        70    79    9.2     SALL683.3              Sal-16  1.6       186    77    20     SALL692.2              Sal-16  2.7       148    63    24     ______________________________________

The relative expression of lipase responded differently to different carbon sources, suggesting that the regulation of the lipase expression was altered in these transformants.

Example 48

Screening for α-Cyclopiazonic Acid Mutants

The pools e1-e26 from the EcoRI library described in Example 5 was used in screening for α-cyclopiazonic acid negative strains.

The spore number in the vials containing the different pools was determined by counting an appropriate dilution in a haemocytometer and a dilution series was constructed in such a way that approximately 30-50 spore derived colonies were present on each 9 cm screening plate. The screening medium was composed per liter of 30 g of mannitol, 10 g of glucose, 10 g of succinic acid, 3 g of Casamino acids, 1 g of KH₂ PO₄, 0.3 g of MgSO₄ --7H₂ O, 0.2 g of FeSO₄ --7H₂ O, 100 μl of Triton X100, and 20 g of Difco Bacto Agar. The pH was adjusted to 5.6 with 14% NH₄ OH before autoclaving. The ferrous ion forms a red complex with α-cyclopiazonic acid. This complex is seen on the reverse side of the colonies.

Approximately 2000-2500 colonies were screened from each pool. Colonies with no red coloration on the reverse side after 7 days incubation at 34° C. were reisolated on the screening medium and incubated for 7 days at 34° C. Ten colonies originating from 6 different pools exhibited non-red reverse coloration and were subsequently inoculated onto Cove-N slants made as follows: 20 ml of Cove salt solution, 4.2 g of NaNO₃, and 60 g of glucose were dissolved in deionised water and the volume made up to 1000 ml. The medium was solidified by 2% Difco Bacto Agar.

Five ml of a spore suspension, made from a Cove slant by adding 10 ml of aqueous 0.01% Tween 80, was inoculated into 500 ml baffled shake flasks containing 100 ml of MDU1B shake flask medium. At the time of inoculation, 1.3 ml of 50% sterile filtered urea was added to each shake flask. The shake flasks were incubated at 250 rpm for 5 days at 34° C.

Ten μl of supernatant from the 5 day old shake flask cultures were applied to the opposite edges of a 20 cm×20 cm TLC plate (Merck Silica Gel 60). The plate was then run for 15 minutes in a chloroform:acetone:propan-2-ol (85:15:20) solvent system (CAP) allowed to dry, turned around and the opposite side was run in a toluene: ethyl acetate: formic acid (5:4:1) solvent system (TEF) for 15 minutes. The plate was allowed to dry thoroughly for 1 hour in a fume hood before spraying with Ehrlich reagent (2 g of 4-dimethylaminobenzaldehyde in 85 ml of 96% ethanol plus 15 ml of 37% hydrochloric acid). α-Cyclopiazonic acid was seen as bluish-violet mushroom shaped spots with a typical low Rf value in the CAP solvent system (a neutral system) whereas the acidic TEF solvent system yielded a typical high Rf value prolonged smear. Solutions of 30, 15, and 7.5 ppm of α-cyclopiazonic acid (Sigma Chemical Co., St. Louis, Mo.) in a 1:1:1 solution of ethanol, methanol, and chloroform were used as standards.

The TLC analysis of ten putative α-cyclopiazonic acid-free transformants showed no sign of α-cyclopiazonic acid. The remaining contents of the shake flasks were filtered through Miracloth and 10 ml of 0.1 M hydrochloric acid were added to 60 ml of each filtrate. The acidified filtrates were then vigorously shaken for 3-5 minutes with 50 ml chloroform. The bottom phases (approximately 25 ml) were each transferred to a 300 ml beaker after phase separation (3 hrs) and the chloroform allowed to evaporate. The residues were each redissolved in 300 μl of chloroform and 10 μl of each concentrate was analyzed by TLC as described above.

None of the 10 strain extracts contained any detectable α-cyclopiazonic acid.

DEPOSIT OF BIOLOGICAL MATERIALS

The following strains have been deposited according to the Budapest Treaty in the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Laboratory, 1815 University Street, Peoria, Ill. 61604, USA.

    ______________________________________                   Accession     Strain        Number       Deposit Date     ______________________________________     E. coli HB101 pDSY109                   NRRL B-21623 September 5, 1996     E. coli DH5α pMT1936                   NRRL B-21832 September 8, 1997     E. coli HB101 pDSY112                   NRRL B-21622 September 5, 1996     E. coli HB101 pDSY138                   NRRL B-21833 September 8, 1997     E. coli DH5α pDSY162                   NRRL B-21831 September 8, 1997     E. coli DH5α pDSY163                   NRRL B-21830 September 8, 1997     E. coli DH5α pSMO1204                   NRRL B-21820 September 8, 1997     E. coli DH5α pSMOH603                   NRRL B-21821 September 8, 1997     E. coli HB101 p4-8.1                   NRRL B-21823 September 8, 1997     E. coli HB101 p7-14.1                   NRRL B-21824 September 8, 1997     E. coli DH5α pHB220                   NRRL B-21825 September 8, 1997     E. coli DH5α pSMO717                   NRRL B-21826 September 8, 1997     E. coli DH5α pSMO321                   NRRL B-21827 September 8, 1997     E. coli DH5α pHowB571                   NRRL B-21828 September 8, 1997     E. coli DH5α pSMO810                   NRRL B-21829 September 8, 1997     ______________________________________

The strains have been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. The deposits represent a substantially pure culture of each deposited strain. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the particular information for which the publication was cited. The publications discussed above are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

It is to be understood that this invention is not limited to the particular methods and compositions described as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials or methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

    __________________________________________________________________________     #             SEQUENCE LISTING     - (1) GENERAL INFORMATION:     -    (iii) NUMBER OF SEQUENCES: 80     - (2) INFORMATION FOR SEQ ID NO:1:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 24 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:     #                24AACC CTGA     - (2) INFORMATION FOR SEQ ID NO:2:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 24 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:     #                24GACC GAGC     - (2) INFORMATION FOR SEQ ID NO:3:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 21 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:     #21                AAAA T     - (2) INFORMATION FOR SEQ ID NO:4:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 34 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:     #        34        GTAT ATAGAGGATT GTGG     - (2) INFORMATION FOR SEQ ID NO:5:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 30 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:     #           30     GCTC CCTTGTGCTG     - (2) INFORMATION FOR SEQ ID NO:6:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 29 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:     #            29    GACC CAGCCGCGC     - (2) INFORMATION FOR SEQ ID NO:7:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 28 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:     #             28   AGGG GGTGGTGG     - (2) INFORMATION FOR SEQ ID NO:8:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 28 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:     #             28   GGAT AGGCCCTA     - (2) INFORMATION FOR SEQ ID NO:9:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 3000 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:     - ACACGGCCTG GACAATGAGA CAACTCTTCT AAAGAGTTCA GATTTAGTAT AT - #ATACTAGC       60     - CAGGACGGAC TTTCCAAATA TTATGTAAAT TAAAGTGTCG TTGTGAAGTG CT - #ACCTATAA      120     - TGCTTAGTAT GTGTATGTCT GGATGCAACG GGCTAATATT ACACATCAAG AA - #GTCCACTC      180     - AATAAGCCAC TGGTGCAAAT TAACTGAATA CACATGTATC TATATCCGGA GC - #AATAAAAA      240     - CTAACGATAA TAATGGCAAG GGTCACTTAT AACAAACTCA TACTGGACAA AA - #AGTATGGA      300     - GAAACAATAG ATTAAAAAGG TCCCGTTCTA TATTTAATCC TCCCGCGACG GA - #GTTTCCTT      360     - ATCCCGATAG ATACGATAAG GATCCAGGTA ACGTCATTCC ATCCCGTGAG AT - #AAAGAGGA      420     - CTCCACCTCA ACAACTAATC ATCAAATCTC CAATCAATAT CAATGAACCC AT - #AACAACTT      480     - AAAAAGCTCT CGGAAAAGTA AAAAGAGCTT CTATCAGCAT ATACACCATG GT - #CCTCGGTG      540     - GATCAAGCGG GTCAAAGGTC ACTCCGTACC TGATCTACCT TGTGTTTATC AC - #AACTTTGG      600     - GGCCACTTCA ATTCGGATAT CATTTGGTAT TACACGGAGC TTGGTCTATG CT - #GGAGGCTT      660     - CAATACATCG GCTGACAATA TATTATGATA GGCTGAGCTC AATGCCCCCC AG - #GCCGTGAT      720     - AACTTGCGAG CGGAAAAGCA TCCATTCGAC AACAACACGG GGTCTCCCGC AA - #TGCATACC      780     - TATGAACCCA TCCCAATTCG GCCTGGTCTC CTCTATATAC ACCCTTGGGG GC - #TTGCTAGG      840     - GGCTCTCCTG GCAGGTCCAG TTTCCACCAA GCATGGCCGC TTGTTCACAC TG - #CGAGCGAC      900     - CACCATCTTC TTCATCCTAG GCCCTATAGC AGAAACATTT GCGCCCAGTA TA - #CCCGTATT      960     - GAGTATGGGT AGGCTTTTAT CTGGTGTTGG TGCGGGCGCT TCTATCGTCG TG - #GGTCCGAT     1020     - ATATATCTCT GAGATTGCTC CTCCTAGTGC TAAGGGTCTT TTCGGCGCTT TT - #ACGCAAAT     1080     - CATGACTAAT GTCGGTATTC TGTTGACACA GTCCCTTGGT TACTTCTTGA GT - #AAAGGAAG     1140     - TATGTGGAGA GTTATACTTG CAATTGCTGG CGCGATCGGA TGCCTTGAGC TT - #CTGGGCCT     1200     - CTTCTTAGTC CCAGAAAGCC CCATCTGGCT TGCAGATCAC CAGAAAGGGA AT - #GTGGCTAG     1260     - ACAGGTGCTA CAACGTATAC GGGGCAGGGA TGCAGACATC GAGCCAGAGG TT - #GAAGGCTG     1320     - GAGAACATCT GCAGCGCCTG AACACAGCTC TGGGGAAGAG CAGTCCCTAC TA - #TCACCCCC     1380     - ATCTGGAAAT ATGCCACCCA AGCAACCTCC GGTTACCATG ATGCGAGCTA TT - #ACTGATTC     1440     - TTTTTACCGC CCTGCCATCA TTGCAGTGGT CGGAGTCATG GTTTCCCAGC AG - #TTCACTGG     1500     - TGTCAACAGC ATCATCATGT ACAGCGTTTC CCTCTTACAG ACCATCCTTC CC - #ACCACTGC     1560     - AGCCCTGTTG TCGGTGATCA TCTCGGCTAT CAATCTTGTA ATCACTCTGG CC - #TGCTCACC     1620     - ACTACCTGAT AAGATTGGTA GACGCTCCTG CCTGCTTCTA AGTATCAGCG GC - #ATGGGTCT     1680     - TAATTCCGTC CTACTGGCGC TAGCCATCTA CTTCAACCTG AAAGCCTTAT CC - #GCCATAGC     1740     - AGTTCTACTT TTCGTTGCTT CTTTCGCCGC CGGTCTAGGC CCAGTCCCCT TC - #ATTTTAGC     1800     - CTCTGAACTC GTTGGCCCGG AGGCTGTCGG CGCCGCACAG AGCTGGGCGC TG - #GGAGCGAA     1860     - CTGGATTGCC ACGTTCATCG TGGCACAATT TTTTCCGATG TTAAACGATT TG - #TTGGGCGG     1920     - ACGAGGCAAG ATCTACTGGA TCTTTGCAGC GATGGCCTGT CTCCTCGGAA GT - #TTCATCTA     1980     - CTGGTGGGTG CCGGAGACCA AGGGGAAGGC TAACGCCGAC GAAGTTTGGG GA - #AGGACCAA     2040     - CCAGCGCAGA CAGGACTAAT TTTTCTGGCC TCTTTGATTT TTTTTTTCTG GG - #CCTTACTC     2100     - TGCTGCCAAC ATTCAGATTA TCAATTAGTA GTCAATCTGT GACTATCCTC TC - #CGAGGGAT     2160     - AGCTTGCAAA GGTGTGACCT CCACAGAGGA ATCTATCGTG TGACAGTATC AA - #AGACAATA     2220     - GAATAGCAAT AATTGGTGCT CTCTACCTAG GAGCATTCGG TGAGAGTGAA AG - #AGTCATAC     2280     - TTGCCTCGGC TTGTTCATCC CAGTCGATCA GTCAGGTTTA GCTCGGCAGT AA - #AAGCAATA     2340     - CCGGTCTACT TCCATCTTCA AACTGTACCG CGGAAACAAA GAGTAAAGGA GG - #GGTCATGA     2400     - TACCTCTAAA TAATGTATAA GTCGTTGACA ATGCTCTTTA TCACCACCCG TT - #GAAGACGT     2460     - CCTTTGATGT CTTGATCATC ACAAGCAGGT TGATCATCTG CGATCGACGT CA - #CTTCGCAC     2520     - CGCACACTGC ATGACAAGTG CGGGGCAGAG GGGCCAACAG GCCCAAAAAT TT - #AGTGATTT     2580     - TGAAGCAATG TTGTTACACC CTTTTACCCC TCAATCCATG ATAAGGGAAA AA - #GAGATGCT     2640     - GAGAGAGGGT CACTGCCACG CTAGACTGGA TTGGTCCGTA TATGCAGGTT TA - #TGCACGCA     2700     - CAGGGGGGCT TCGTTCTTCT TGGCTATGCA CTATGGATTA GTAGGGTGTA TT - #CAACCACG     2760     - TAGATAGATT GGCGTGTCCG GTGCAGGATA TGTAGAAGAC AATGAGGTTC GG - #GCTTTCGG     2820     - AAATGAGGAA AGAATGTTGG ACAGATGAAA AACGGTACTG CTGTTGCAAG GG - #GGCGCTGT     2880     - TTGAGATATT TTAAGTGCCT GTCATGTAAT TTTGCAACGG TGAGACATTT AT - #CTAGGGTA     2940     - AAATCCAAGA AGAACCTAGG GAAGAGTAAA GCCACAACGA AGATTACGTG AG - #AGGAAGAG     3000     - (2) INFORMATION FOR SEQ ID NO:10:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 488 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: None     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:     - Met Val Leu Gly Gly Ser Ser Gly Ser Lys Va - #l Thr Pro Tyr Leu Ile     #                15     - Tyr Leu Val Phe Ile Thr Thr Leu Gly Pro Le - #u Gln Phe Gly Tyr His     #            30     - Leu Ala Glu Leu Asn Ala Pro Gln Ala Val Il - #e Thr Cys Glu Arg Lys     #        45     - Ser Ile His Ser Thr Thr Thr Arg Gly Leu Pr - #o Gln Cys Ile Pro Met     #    60     - Asn Pro Ser Gln Phe Gly Leu Val Ser Ser Il - #e Tyr Thr Leu Gly Gly     #80     - Leu Leu Gly Ala Leu Leu Ala Gly Pro Val Se - #r Thr Lys His Gly Arg     #                95     - Leu Phe Thr Leu Arg Ala Thr Thr Ile Phe Ph - #e Ile Leu Gly Pro Ile     #           110     - Ala Glu Thr Phe Ala Pro Ser Ile Pro Val Le - #u Ser Met Gly Arg Leu     #       125     - Leu Ser Gly Val Gly Ala Gly Ala Ser Ile Va - #l Val Gly Pro Ile Tyr     #   140     - Ile Ser Glu Ile Ala Pro Pro Ser Ala Lys Gl - #y Leu Phe Gly Ala Phe     145                 1 - #50                 1 - #55                 1 -     #60     - Thr Gln Ile Met Thr Asn Val Gly Ile Leu Le - #u Thr Gln Ser Leu Gly     #               175     - Tyr Phe Leu Ser Lys Gly Ser Met Trp Arg Va - #l Ile Leu Ala Ile Ala     #           190     - Gly Ala Ile Gly Cys Leu Glu Leu Leu Gly Le - #u Phe Leu Val Pro Glu     #       205     - Ser Pro Ile Trp Leu Ala Asp His Gln Lys Gl - #y Asn Val Ala Arg Gln     #   220     - Val Leu Gln Arg Ile Arg Gly Arg Asp Ala As - #p Ile Glu Pro Glu Val     225                 2 - #30                 2 - #35                 2 -     #40     - Glu Gly Trp Arg Thr Ser Ala Ala Pro Glu Hi - #s Ser Ser Gly Glu Glu     #               255     - Gln Ser Leu Leu Ser Pro Pro Ser Gly Asn Me - #t Pro Pro Lys Gln Pro     #           270     - Pro Val Thr Met Met Arg Ala Ile Thr Asp Se - #r Phe Tyr Arg Pro Ala     #       285     - Ile Ile Ala Val Val Gly Val Met Val Ser Gl - #n Gln Phe Thr Gly Val     #   300     - Asn Ser Ile Ile Met Tyr Ser Val Ser Leu Le - #u Gln Thr Ile Leu Pro     305                 3 - #10                 3 - #15                 3 -     #20     - Thr Thr Ala Ala Leu Leu Ser Val Ile Ile Se - #r Ala Ile Asn Leu Val     #               335     - Ile Thr Leu Ala Cys Ser Pro Leu Pro Asp Ly - #s Ile Gly Arg Arg Ser     #           350     - Cys Leu Leu Leu Ser Ile Ser Gly Met Gly Le - #u Asn Ser Val Leu Leu     #       365     - Ala Leu Ala Ile Tyr Phe Asn Leu Lys Ala Le - #u Ser Ala Ile Ala Val     #   380     - Leu Leu Phe Val Ala Ser Phe Ala Ala Gly Le - #u Gly Pro Val Pro Phe     385                 3 - #90                 3 - #95                 4 -     #00     - Ile Leu Ala Ser Glu Leu Val Gly Pro Glu Al - #a Val Gly Ala Ala Gln     #               415     - Ser Trp Ala Leu Gly Ala Asn Trp Ile Ala Th - #r Phe Ile Val Ala Gln     #           430     - Phe Phe Pro Met Leu Asn Asp Leu Leu Gly Gl - #y Arg Gly Lys Ile Tyr     #       445     - Trp Ile Phe Ala Ala Met Ala Cys Leu Leu Gl - #y Ser Phe Ile Tyr Trp     #   460     - Trp Val Pro Glu Thr Lys Gly Lys Ala Asn Al - #a Asp Glu Val Trp Gly     465                 4 - #70                 4 - #75                 4 -     #80     - Arg Thr Asn Gln Arg Arg Gln Asp                     485     - (2) INFORMATION FOR SEQ ID NO:11:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 488 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: None     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:     - Met Ala Glu Thr Glu Arg Leu Met Pro Asn Gl - #y Gly Ser Arg Glu Thr     #                15     - Lys Pro Leu Ile Thr Gly His Leu Ile Leu Gl - #y Thr Ile Val Ala Cys     #            30     - Leu Gly Ser Ile Gln Tyr Gly Tyr His Ile Al - #a Glu Leu Asn Ala Pro     #        45     - Gln Glu Phe Leu Ser Cys Ser Arg Phe Glu Al - #a Pro Asp Glu Asn Ile     #    60     - Ser Tyr Asp Asp Thr Trp Val Gly Gln His Gl - #y Leu Lys Gln Cys Ile     #80     - Ala Leu Thr Asp Ser Gln Tyr Gly Ala Ile Th - #r Ser Ile Phe Ser Ile     #                95     - Gly Gly Leu Phe Gly Ser Tyr Tyr Ala Gly As - #n Trp Ala Asn Arg Tyr     #           110     - Gly Arg Lys Tyr Val Ser Met Gly Ala Ser Al - #a Met Cys Met Val Ser     #       125     - Ser Leu Leu Leu Phe Phe Ser Asn Ser Tyr Le - #u Gln Leu Leu Phe Gly     #   140     - Arg Phe Leu Val Gly Met Ser Cys Gly Thr Al - #a Ile Val Ile Thr Pro     145                 1 - #50                 1 - #55                 1 -     #60     - Leu Phe Ile Asn Glu Ile Ala Pro Val Glu Tr - #p Arg Gly Ala Met Gly     #               175     - Ser Met Asn Gln Val Ser Ile Asn Leu Gly Il - #e Leu Leu Thr Gln Thr     #           190     - Leu Ala Leu Lys Tyr Ala Asp Ser Tyr Asn Tr - #p Arg Trp Leu Leu Phe     #       205     - Ser Gly Ser Val Ile Ala Val Ala Asn Ile Le - #u Ala Trp Leu Lys Val     #   220     - Asp Glu Ser Pro Arg Trp Leu Val Ser His Gl - #y Phe Val Ser Glu Ala     225                 2 - #30                 2 - #35                 2 -     #40     - Glu Thr Ala Leu Phe Lys Leu Arg Pro Gly Th - #r Tyr Gln Gln Ala Lys     #               255     - Gln Glu Ile Gln Asp Trp Gln Arg Ser His Gl - #y His Asn Arg Asp Pro     #           270     - Glu Ser Ser Glu Glu Thr His Ser Gly Pro Th - #r Leu Trp Gln Tyr Val     #       285     - Thr Asp Pro Ser Tyr Lys Lys Pro Arg Thr Va - #l Ile Leu Ala Ile Leu     #   300     - Ser Cys Gln Gln Phe Cys Gly Ile Asn Ser Il - #e Ile Phe Tyr Gly Val     305                 3 - #10                 3 - #15                 3 -     #20     - Lys Val Ile Gly Lys Ile Leu Pro Asp Tyr Se - #r Ile Gln Val Asn Phe     #               335     - Ala Ile Ser Ile Leu Asn Val Val Val Thr Le - #u Ala Ala Ser Ala Ile     #           350     - Ile Asp His Val Gly Arg Arg Pro Leu Leu Le - #u Ala Ser Thr Thr Val     #       365     - Met Thr Ala Met Ser Leu Leu Ile Ser Val Gl - #y Leu Thr Leu Ser Val     #   380     - Ser Phe Leu Leu Val Thr Ala Thr Phe Val Ty - #r Ile Ala Ala Phe Ala     385                 3 - #90                 3 - #95                 4 -     #00     - Ile Gly Leu Gly Pro Ile Pro Phe Leu Ile Il - #e Gly Glu Leu Ser Tyr     #               415     - Pro Gln Asp Ala Ala Thr Ala Gln Ser Phe Gl - #y Thr Val Cys Asn Trp     #           430     - Leu Ala Thr Phe Ile Val Gly Tyr Leu Phe Pr - #o Ile Gly His Gly Leu     #       445     - Met Gly Gly Tyr Val Phe Ala Ile Phe Ala Al - #a Ile Ala Ala Met Phe     #   460     - Ala Thr Tyr Val Tyr Lys Arg Val Pro Glu Th - #r Lys Gly Lys Thr Thr     465                 4 - #70                 4 - #75                 4 -     #80     - Tyr Ser Glu Val Trp Ala Gly Tyr                     485     - (2) INFORMATION FOR SEQ ID NO:12:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 524 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: None     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:     - Met Thr Glu Asp Lys Val Thr Gly Thr Leu Va - #l Phe Thr Val Ile Thr     #                15     - Ala Val Leu Gly Ser Phe Gln Phe Gly Tyr As - #p Ile Gly Val Ile Asn     #            30     - Ala Pro Gln Gln Val Ile Ile Ser His Tyr Ar - #g His Val Leu Gly Val     #        45     - Pro Leu Asp Asp Arg Lys Ala Ile Asn Asn Ty - #r Val Ile Asn Ser Thr     #    60     - Asp Glu Leu Pro Thr Ile Ser Tyr Ser Met As - #n Pro Lys Pro Thr Pro     #80     - Trp Ala Glu Glu Glu Thr Val Ala Ala Ala Gl - #n Leu Ile Thr Met Leu     #                95     - Trp Ser Leu Ser Val Ser Ser Phe Ala Val Gl - #y Gly Met Thr Ala Ser     #           110     - Phe Phe Gly Gly Trp Leu Gly Asp Thr Leu Gl - #y Arg Ile Lys Ala Met     #       125     - Leu Val Ala Asn Ile Leu Ser Leu Val Gly Al - #a Leu Leu Met Gly Phe     #   140     - Ser Lys Leu Gly Pro Ser His Ile Leu Ile Il - #e Ala Gly Arg Ser Ile     145                 1 - #50                 1 - #55                 1 -     #60     - Ser Gly Leu Tyr Cys Gly Leu Ile Ser Gly Le - #u Val Pro Met Tyr Ile     #               175     - Gly Glu Ile Ala Pro Thr Ala Leu Arg Gly Al - #a Leu Gly Thr Phe His     #           190     - Gln Leu Ala Ile Val Thr Gly Ile Leu Ile Se - #r Gln Ile Ile Gly Leu     #       205     - Glu Phe Ile Leu Gly Asn Tyr Asp Leu Trp Hi - #s Ile Leu Leu Gly Leu     #   220     - Ser Gly Val Arg Ala Ile Leu Gln Ser Leu Le - #u Leu Phe Phe Cys Pro     225                 2 - #30                 2 - #35                 2 -     #40     - Glu Ser Pro Arg Tyr Leu Tyr Ile Lys Leu As - #p Glu Glu Val Lys Ala     #               255     - Lys Gln Ser Leu Lys Arg Leu Arg Gly Tyr As - #p Asp Val Thr Lys Asp     #           270     - Ile Asn Glu Met Arg Lys Glu Arg Glu Glu Al - #a Ser Ser Glu Gln Lys     #       285     - Val Ser Ile Ile Gln Leu Phe Thr Asn Ser Se - #r Tyr Arg Gln Pro Ile     #   300     - Leu Val Ala Leu Met Leu His Val Ala Gln Gl - #n Phe Ser Gly Ile Asn     305                 3 - #10                 3 - #15                 3 -     #20     - Gly Ile Phe Tyr Tyr Ser Thr Ser Ile Phe Gl - #n Thr Ala Gly Ile Ser     #               335     - Lys Pro Val Tyr Ala Thr Ile Gly Val Gly Al - #a Val Asn Met Val Phe     #           350     - Thr Ala Val Ser Val Phe Leu Val Glu Lys Al - #a Gly Arg Arg Ser Leu     #       365     - Phe Leu Ile Gly Met Ser Gly Met Phe Val Cy - #s Ala Ile Phe Met Ser     #   380     - Val Gly Leu Val Leu Leu Asn Lys Phe Ser Tr - #p Met Ser Tyr Val Ser     385                 3 - #90                 3 - #95                 4 -     #00     - Met Ile Ala Ile Phe Leu Phe Val Ser Phe Ph - #e Glu Ile Gly Pro Gly     #               415     - Pro Ile Pro Trp Phe Met Val Ala Glu Phe Ph - #e Ser Gln Gly Pro Arg     #           430     - Pro Ala Ala Leu Ala Ile Ala Ala Phe Ser As - #n Trp Thr Cys Asn Phe     #       445     - Ile Val Ala Leu Cys Phe Gln Tyr Ile Ala As - #p Phe Cys Gly Pro Tyr     #   460     - Val Phe Phe Leu Phe Ala Gly Val Leu Leu Al - #a Phe Thr Leu Phe Thr     465                 4 - #70                 4 - #75                 4 -     #80     - Phe Phe Lys Val Pro Glu Thr Lys Gly Lys Se - #r Phe Glu Glu Ile Ala     #               495     - Ala Glu Phe Gln Lys Lys Ser Gly Ser Ala Hi - #s Arg Pro Lys Ala Ala     #           510     - Val Glu Met Lys Phe Leu Gly Ala Thr Glu Th - #r Val     #       520     - (2) INFORMATION FOR SEQ ID NO:13:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 584 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: None     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:     - Met Gly Ile His Ile Pro Tyr Leu Thr Ser Ly - #s Thr Ser Gln Ser Asn     #                15     - Val Gly Asp Ala Val Gly Asn Ala Asp Ser Va - #l Glu Phe Asn Ser Glu     #            30     - His Asp Ser Pro Ser Lys Arg Gly Lys Ile Hi - #s Ile Glu Ser His Glu     #        45     - Ile Gln Arg Ala Pro Ala Ser Asp Asp Glu As - #p Arg Ile Gln Ile Lys     #    60     - Pro Val Asn Asp Glu Asp Asp Thr Ser Val Me - #t Ile Thr Phe Asn Gln     #80     - Ser Leu Ser Pro Phe Ile Ile Thr Leu Thr Ph - #e Val Ala Ser Ile Ser     #                95     - Gly Phe Met Phe Gly Tyr Asp Thr Gly Tyr Il - #e Ser Ser Ala Leu Ile     #           110     - Ser Ile Gly Thr Asp Leu Asp His Lys Val Le - #u Thr Tyr Gly Glu Lys     #       125     - Glu Ile Val Thr Ala Ala Thr Ser Leu Gly Al - #a Leu Ile Thr Ser Ile     #   140     - Phe Ala Gly Thr Ala Ala Asp Ile Phe Gly Ar - #g Lys Arg Cys Leu Met     145                 1 - #50                 1 - #55                 1 -     #60     - Gly Ser Asn Leu Met Phe Val Ile Gly Ala Il - #e Leu Gln Val Ser Ala     #               175     - His Thr Phe Trp Gln Met Ala Val Gly Arg Le - #u Ile Met Gly Phe Gly     #           190     - Val Gly Ile Gly Ser Leu Ile Ala Pro Leu Ph - #e Ile Ser Glu Ile Ala     #       205     - Pro Lys Met Ile Arg Gly Arg Leu Thr Val Il - #e Asn Ser Leu Trp Leu     #   220     - Thr Gly Gly Gln Leu Val Ala Tyr Gly Cys Gl - #y Ala Gly Leu Asn Tyr     225                 2 - #30                 2 - #35                 2 -     #40     - Val Asn Asn Gly Trp Arg Ile Leu Val Gly Le - #u Ser Leu Ile Pro Thr     #               255     - Ala Val Gln Phe Thr Cys Leu Cys Phe Leu Pr - #o Asp Thr Pro Arg Tyr     #           270     - Tyr Val Met Lys Gly Asp Leu Ala Arg Ala Th - #r Glu Val Leu Lys Arg     #       285     - Ser Tyr Thr Asp Thr Ser Glu Glu Ile Ile Gl - #u Arg Lys Val Glu Glu     #   300     - Leu Val Thr Leu Asn Gln Ser Ile Pro Gly Ly - #s Asn Val Pro Glu Lys     305                 3 - #10                 3 - #15                 3 -     #20     - Val Trp Asn Thr Ile Lys Glu Leu His Thr Va - #l Pro Ser Asn Leu Arg     #               335     - Ala Leu Ile Ile Gly Cys Gly Leu Gln Ala Il - #e Gln Gln Phe Thr Gly     #           350     - Trp Asn Ser Leu Met Tyr Phe Ser Gly Thr Il - #e Phe Glu Thr Val Gly     #       365     - Phe Lys Asn Ser Ser Ala Val Ser Ile Ile Va - #l Ser Gly Thr Asn Phe     #   380     - Ile Phe Thr Leu Val Ala Phe Phe Ser Ile As - #p Lys Ile Gly Arg Arg     385                 3 - #90                 3 - #95                 4 -     #00     - Thr Ile Leu Leu Ile Gly Leu Pro Gly Met Th - #r Met Ala Leu Val Val     #               415     - Cys Ser Ile Ala Phe His Phe Leu Gly Ile Ly - #s Phe Asp Gly Ala Val     #           430     - Ala Val Val Val Ser Ser Gly Phe Ser Ser Tr - #p Gly Ile Val Ile Ile     #       445     - Val Phe Ile Ile Val Phe Ala Ala Phe Tyr Al - #a Leu Gly Ile Gly Thr     #   460     - Val Pro Trp Gln Gln Ser Glu Leu Phe Pro Gl - #n Asn Val Arg Gly Ile     465                 4 - #70                 4 - #75                 4 -     #80     - Gly Thr Ser Tyr Ala Thr Ala Thr Asn Trp Al - #a Gly Ser Leu Val Ile     #               495     - Ala Ser Thr Phe Leu Thr Met Leu Gln Asn Il - #e Thr Pro Ala Gly Thr     #           510     - Phe Ala Phe Phe Ala Gly Leu Ser Cys Leu Se - #r Thr Ile Phe Cys Tyr     #       525     - Phe Cys Tyr Pro Glu Leu Ser Gly Leu Glu Le - #u Glu Glu Val Gln Thr     #   540     - Ile Leu Lys Asp Gly Phe Asn Ile Lys Ala Se - #r Lys Ala Leu Ala Lys     545                 5 - #50                 5 - #55                 5 -     #60     - Lys Arg Lys Gln Gln Val Ala Arg Val His Gl - #u Leu Lys Tyr Glu Pro     #               575     - Thr Gln Glu Ile Ile Glu Asp Ile                 580     - (2) INFORMATION FOR SEQ ID NO:14:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 21 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:     #21                CCAA G     - (2) INFORMATION FOR SEQ ID NO:15:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 21 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:     #21                GGAT T     - (2) INFORMATION FOR SEQ ID NO:16:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 4700 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:     - ACAGCGACTG GGATGGTGAA TATCTGAGCG ATAGCCCCCG ACACAGCACC AA - #GGGTAAGC       60     - TCCATAGCGG TTCCAGGTGG CTTAGATACG CTCTTCGATG CCATATAAAG GC - #TTCTAACC      120     - ACGCTGTACC AGTAGAAGTA CGCAAAGTTG GTCGAGGCCA CGCCAAGCAA AG - #AACCAACC      180     - ATCCCGGAAT ATAAACCTTC AATTCCCTCT TTCTCCACAA TCTTGTTGAT GG - #CATCTAGG      240     - GTCGACTCGT AATGTACTAC ATCTCCGCTT TTCGATTCAG GTGCGTTCTT CA - #CTTGGACT      300     - TGAAGTTTGG TTTTGACACT AAAGATTAGG AACGAGTCAG TCTCAGTCTA CA - #CCACTAAG      360     - CCAACTGGCA AGGCTATGTA CGCACAGGTC CAGTGGATAG ACGATAGCAT TT - #GCGAGAAC      420     - AGCACCAGTT GCACCTGCGA CAGCACTACC CCAAGGGGAG AGCGCGGGTT TC - #GATTGGCC      480     - GGCCATTATG CAGGATGAGC TAAAGTGCCT CTGCCAATTC CGTCAGAAAG AA - #TAGTATAA      540     - GAGCACAAAT ACTGGTAAAA CCAAGACCGG CGAATGAGGC AGGACTCTGT GC - #GATTCGGG      600     - GGGTTTAGCG TTCGGCTTGA AGGCTTACCG GATCGACTGA TAGAAAAGTT GA - #ATGCCGAG      660     - TAAGGTGAAA AGACCCCAGC TCCCAAGCAG CAACAGTAAG TGGAAGAGCT TA - #AGGATAGA      720     - AAAAATAAAT TAGGATTAAG AAAAAAAGAA GAACCTCAAG ACTGGTCACA CA - #GTCCCGGC      780     - ATCCTGAACG TAAAATGCGG GAAGGATAGA GTCGGCAGGG CCAGGGCAGT TG - #CACCTCGG      840     - CGCTCTGGTT TGCGCATGAC GAAATGAGCC GAGGTTCGTT TTTTGGAGGC CA - #ATTTCTGA      900     - ACACCGACCT TCGAATTCCC GTTCCTCCCC ACCGACACGC TAGTGAATGA TC - #CAGCAAGC      960     - ATACTTGGTG TTGTTTTGAC CTCATTCCAC TGCGTGTGAA TTAGCATTAA TT - #TAAGTTTA     1020     - TGATTAACAG TCAATTGCTA TACGCGAAAA TCATCATCGT CTTGATTGGC CC - #TTCATAAA     1080     - ACTTGACAAG GAAGTTTGAT CGACCTCGGA TGTCGCGCTT TCGGAAATTT CA - #CGGAGCCC     1140     - TTCGGACGGG TCACAAGCAA GGTGTCTGAC TGTCTCGTTT AGTCGGATAG AC - #GCTAGTTG     1200     - AACTGTTATG CCTATCGCGG GGAAGATCTC GGAGTGTCAC GGTGTTTGAA GA - #TCCCAGGC     1260     - GCTCGTCAAA ATACTGCCCG GCCTGCCAGT ATGTCTAGAC CGAACGCCTC AG - #CCCAGAAG     1320     - TCCTTTATAA CTCAGGCACT GGTACTTGAC CCTTTTTTTT TATGGTTTTT TG - #TTTCTTTC     1380     - TTGTTACACC TTATTTTTCT TCTTCTCGTT TTTTGTAGAT AATACTGACC AC - #TGGCTAGA     1440     - AAGCCGAGCG GGATGTATCG TCCGCCACTT CTCAAAGGCA AGCTTTAGAA GC - #TGCCATTG     1500     - ATGCTGCTGA ACACTATATG AAAGCCTTAA ATCTGGCATC TGTTCAGAAA GA - #CAAACATG     1560     - CATTGGATGC AAAGTGTAAA GAATGGCTCA CAAGAGCGGA AAAGATCAAA GA - #ATCTAAGG     1620     - ACTGGCAAGC TGCTGCCCGT TTCCATGACA AAACTGTTCC AGAGCCACGG TT - #GCCTGTAT     1680     - CTACTCGTAA GCTCACCACA CGGGAGGAGA TCATTCTGCT AGAGGGAGCC AA - #GTTGAATG     1740     - GCTTCATATT CCCTCCATGG TCCACCTCCC CAGGCTCTGA CGAGTTCAAA CG - #AGAGGATG     1800     - GTGAATCCCC GTTTACGTAA GTTCTGGTGG TCTGCATCGT CAATGTTGCA TG - #TATACCCA     1860     - GATGACTGCT GGATATTCTA ACCGATAACA GCGACAAACC CGATCTTCAT CT - #ATCTTATC     1920     - CTCAAAGGAA AGTTTTTGAT GGCTGGAAAC GACCTTCCGA GCTTCTCGCG AA - #AGACACGG     1980     - AAGATGTGTA CACAAAGGTG GTTCCTGTGA TGTCTGTTCC AGGAAAGACA GA - #TCTAGTCC     2040     - AGGATATGCT GACGGACTGT TCTGTCGTTG CTAGCCTTTG TGCTACTACG TC - #AATGCTAG     2100     - AACGCGGCCA GTGTACTGTA AGAAGATTGA TCCCTTCCGG CTGACCTGCA TG - #GTTCGCTG     2160     - TGACTAATAG GTGTAGCATT TTCTTCCAAT GATATACCCT AGCCGGGGGA GC - #TCTCAGCC     2220     - TTCACCGTCA GGCAAGTATA TATTTCGCTT TTATTTCAAT GGGTGCTTCC GG - #AAAGTCAT     2280     - CATTGACGAC CGTTTGCCAT CGTCTAAGAC ATCAAGATCA CTCCACGTGA TC - #GACCGGAA     2340     - AAATCCCAAT TTCCTTTGGC CGGCGCTCGT AGAGAAGGCG TATTTGAAAT TG - #CGCGGAGG     2400     - CTATGATTTT CCCGGAAGCA ATTCCGGGAC AGATCTCTGG GTGCTGACAG GT - #TGGATTCC     2460     - CGAGCAAGTC TTTCTCCATA ATGACGATGT GACTGGCGAC CAGCTCTGGA AG - #CGACTTTA     2520     - CAGATCCTTT CACCAAGGAG ATGTTCTCTT GACTATAGGT ACCGGTGAAC TC - #ACTGAGAG     2580     - GGAACAAAGA GAACTAGGCC TCGTGAGTGA GCATGATTAT GCTATTCTGG AT - #ATGAAGGA     2640     - ATCTAAAGGT CGCCGACAAT TACTCGTGAA AAACCCTTGG GCTGGAGCAG AT - #ACTGCCCC     2700     - CGGCGACAAT GGAAGCCTCT CTGCATCGCA GGATTTACCC CATAACCCGC CC - #TCATTTGA     2760     - GCCGGGTACC TTTTGGATGG ATTGCGAAAA GCTGCTTCAA CATTTTGAAA AC - #CTCTATTT     2820     - GAATTGGAAC CCTGAGATTT TCAAATACCG CGAAGACGTC CACTTTACGT GG - #GACCTCAA     2880     - CAACGGGAGA GGTGTAGCCG GCTGTTTTGT GAATAACCCG CAGTTCGCAG TG - #TCAACCGA     2940     - GAACGGTGGG ATTGTCTGGT TACTTCTAGG CAAGCATTTC AGAACAACAG GG - #CAGCCGGA     3000     - ACGACCTCTT GACGAATACC AAGCGAATGA GGAGTCGGCT TTTATAAGCA TA - #TATGTCTT     3060     - TAACGCAGAT GGCAAACGGG TCTCTTTGAG TGATGGGGCT CTACATCGTG GC - #CCCTATGT     3120     - GGATTCCCCT AATACGCTCA TGAGGTTAGA GATGCCCCCC AGAACAACAT AC - #ACAGTCGT     3180     - GGTCTCCGAG CAATCACTGC CATCTTTGAA TCAAAACTTT ACTTTGTCTG CC - #TTCTCTAC     3240     - CTGCCCTGTA CGGATGGCAA AAGCCCAAGA TAAATACATG TGTGTCAGGA AG - #ATTCAAGG     3300     - GTCTTGGACA CCTTCGACGG CAGGTGGGAA TGCCGAATCT TCTCGATATC CA - #CTCAACCC     3360     - CCAATTTAGG TTGGAGATAG AGAATGACAC AGATGTTTCA CTCCTGCTGG AA - #TGCCCAAA     3420     - CACGGAACTC GCGACCCATG TTAAGTTATT CTGGTCCAAT GGAAATCGTG TG - #TCGCGAGT     3480     - ACGCAGTCGC GACATAATCG CTGATAGTGG TGACTATCGC CGTGGTGGCT CC - #CTTGTGGA     3540     - AAAGAAGGCT CTGGAACCGG GCTCATATAC AATCGTCTGT TCCACATTCG CG - #CCGGATCA     3600     - ACTTGGCCGA TTCACGCTCT GGGTATCCTC CTTAGTTCCT TGCAAGACGA GC - #CCGCTCCC     3660     - ACCAGAGGCA GCAGGTCGAC GAACGGTCAT TTCAGATATT GGCGTACTGC CT - #CCCGGGAG     3720     - AGACCGAATG TTAGCTTCTC TGCAAGTGCC GCGGCTTACG AGGATCAAGC TC - #ATCACCCG     3780     - AAGTAGGCAA TCCATCATCG GGAGCCATCC TGTTGGACCC TCGCCCGTTT TA - #ATGACAGT     3840     - GGAGCTCGGG CAAGGGCCAT ACAAACAGAT CCTGGCGACT TCGGAAGATG GA - #ACTCACAG     3900     - TGATGCTGTA TCGGGGGTAC GTGTTGAGGA CTTTGACTTG CAGCCTGGGC TA - #GAGGAGAG     3960     - TGGTGGTATT TGGATTGTTA TTGAGAGGAT TGGGGGTCCT GGAGGGCAGG TA - #GAGGACCA     4020     - CTTTGAGGTG GAAGCTTTGG CTGAAGAGAG GGTTGAGATT GGGGAGTGGA TA - #CTTGAAGA     4080     - TGCTTGATCT CTTATCCTGC AGAAGCTCTG AAGCTCTGGA CGGTCTTAGT TG - #AGCTTTTT     4140     - TGATCGTCGT TGTGATTAGC ACGTTAGAGT AGAAGAGCGG AAACAATGAT AG - #ACATGAAT     4200     - TTCTCTTATT GTCTCTATTG GCCAGAAGAG AAAGAAGGCA TTAAATCAAT AC - #ATTAAAAG     4260     - CAAGAGTTTA TCTATAGATA CCGAGCAGCC TCAGGATTTG AGCTGAGGTT TG - #TCGCGATC     4320     - GCGACCGCCA AAATGGAGTT AGCTTGCTTT ACTCCGCATA AATTAAATCC TC - #GGCTCGGG     4380     - GCCCGAATTC CCCTCCCGAG TGCTTTCAAC GTCATCCCGT TTGCTGTGTG CA - #TTGTGCCT     4440     - CCCCACCTTT AACAATTGGA GCTCTGTCCA AGGACAAATC CTTATTCTCG CG - #GCCTCCAT     4500     - GGCGACTAAT ATTCCTGCTG CTCTGAAGTC TGCGGACATT GGGCGCTTTG CC - #GTCAGAGC     4560     - AGCTCAGCTT GAACGTGTAA AGCCCGTGGT CGCCTACTGG TGTGAGTATT GT - #GTGATTAT     4620     - ACCCAGTACG AACTACGAGC TGATGAGCGG CCTCTGCTGA TCGCAGGCAA CT - #TCTGGATC     4680     #                 470 - #0     - (2) INFORMATION FOR SEQ ID NO:17:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 854 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: None     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:     - Met Ser Arg Pro Asn Ala Ser Ala Gln Lys Se - #r Phe Ile Thr Gln Ala     #                15     - Leu Lys Ala Glu Arg Asp Val Ser Ser Ala Th - #r Ser Gln Arg Gln Ala     #            30     - Leu Glu Ala Ala Ile Asp Ala Ala Glu His Ty - #r Met Lys Ala Leu Asn     #        45     - Leu Ala Ser Val Gln Lys Asp Lys His Ala Le - #u Asp Ala Lys Cys Lys     #    60     - Glu Trp Leu Thr Arg Ala Glu Lys Ile Lys Gl - #u Ser Lys Asp Trp Gln     #80     - Ala Ala Ala Arg Phe His Asp Lys Thr Val Pr - #o Glu Pro Arg Leu Pro     #                95     - Val Ser Thr Arg Lys Leu Thr Thr Arg Glu Gl - #u Ile Ile Leu Leu Glu     #           110     - Gly Ala Lys Leu Asn Gly Phe Ile Phe Pro Pr - #o Trp Ser Thr Ser Pro     #       125     - Gly Ser Asp Glu Phe Lys Arg Glu Asp Gly Gl - #u Ser Pro Phe Thr Asp     #   140     - Lys Pro Asp Leu His Leu Ser Tyr Pro Gln Ar - #g Lys Val Phe Asp Gly     145                 1 - #50                 1 - #55                 1 -     #60     - Trp Lys Arg Pro Ser Glu Leu Leu Ala Lys As - #p Thr Glu Asp Val Tyr     #               175     - Thr Lys Val Val Pro Val Met Ser Val Pro Gl - #y Lys Thr Asp Leu Val     #           190     - Gln Asp Met Leu Thr Asp Cys Ser Val Val Al - #a Ser Leu Cys Ala Thr     #       205     - Thr Ser Met Leu Glu Arg Gly Gln Cys Thr Hi - #s Phe Leu Pro Met Ile     #   220     - Tyr Pro Ser Arg Gly Ser Ser Gln Pro Ser Pr - #o Ser Gly Lys Tyr Ile     225                 2 - #30                 2 - #35                 2 -     #40     - Phe Arg Phe Tyr Phe Asn Gly Cys Phe Arg Ly - #s Val Ile Ile Asp Asp     #               255     - Arg Leu Pro Ser Ser Lys Thr Ser Arg Ser Le - #u His Val Ile Asp Arg     #           270     - Lys Asn Pro Asn Phe Leu Trp Pro Ala Leu Va - #l Glu Lys Ala Tyr Leu     #       285     - Lys Leu Arg Gly Gly Tyr Asp Phe Pro Gly Se - #r Asn Ser Gly Thr Asp     #   300     - Leu Trp Val Leu Thr Gly Trp Ile Pro Glu Gl - #n Val Phe Leu His Asn     305                 3 - #10                 3 - #15                 3 -     #20     - Asp Asp Val Thr Gly Asp Gln Leu Trp Lys Ar - #g Leu Tyr Arg Ser Phe     #               335     - His Gln Gly Asp Val Leu Leu Thr Ile Gly Th - #r Gly Glu Leu Thr Glu     #           350     - Arg Glu Gln Arg Glu Leu Gly Leu Val Ser Gl - #u His Asp Tyr Ala Ile     #       365     - Leu Asp Met Lys Glu Ser Lys Gly Arg Arg Gl - #n Leu Leu Val Lys Asn     #   380     - Pro Trp Ala Gly Ala Asp Thr Ala Pro Gly As - #p Asn Gly Ser Leu Ser     385                 3 - #90                 3 - #95                 4 -     #00     - Ala Ser Gln Asp Leu Pro His Asn Pro Pro Se - #r Phe Glu Pro Gly Thr     #               415     - Phe Trp Met Asp Cys Glu Lys Leu Leu Gln Hi - #s Phe Glu Asn Leu Tyr     #           430     - Leu Asn Trp Asn Pro Glu Ile Phe Lys Tyr Ar - #g Glu Asp Val His Phe     #       445     - Thr Trp Asp Leu Asn Asn Gly Arg Gly Val Al - #a Gly Cys Phe Val Asn     #   460     - Asn Pro Gln Phe Ala Val Ser Thr Glu Asn Gl - #y Gly Ile Val Trp Leu     465                 4 - #70                 4 - #75                 4 -     #80     - Leu Leu Gly Lys His Phe Arg Thr Thr Gly Gl - #n Pro Glu Arg Pro Leu     #               495     - Asp Glu Tyr Gln Ala Asn Glu Glu Ser Ala Ph - #e Ile Ser Ile Tyr Val     #           510     - Phe Asn Ala Asp Gly Lys Arg Val Ser Leu Se - #r Asp Gly Ala Leu His     #       525     - Arg Gly Pro Tyr Val Asp Ser Pro Asn Thr Le - #u Met Arg Leu Glu Met     #   540     - Pro Pro Arg Thr Thr Tyr Thr Val Val Val Se - #r Glu Gln Ser Leu Pro     545                 5 - #50                 5 - #55                 5 -     #60     - Ser Leu Asn Gln Asn Phe Thr Leu Ser Ala Ph - #e Ser Thr Cys Pro Val     #               575     - Arg Met Ala Lys Ala Gln Asp Lys Tyr Met Cy - #s Val Arg Lys Ile Gln     #           590     - Gly Ser Trp Thr Pro Ser Thr Ala Gly Gly As - #n Ala Glu Ser Ser Arg     #       605     - Tyr Pro Leu Asn Pro Gln Phe Arg Leu Glu Il - #e Glu Asn Asp Thr Asp     #   620     - Val Ser Leu Leu Leu Glu Cys Pro Asn Thr Gl - #u Leu Ala Thr His Val     625                 6 - #30                 6 - #35                 6 -     #40     - Lys Leu Phe Trp Ser Asn Gly Asn Arg Val Se - #r Arg Val Arg Ser Arg     #               655     - Asp Ile Ile Ala Asp Ser Gly Asp Tyr Arg Ar - #g Gly Gly Ser Leu Val     #           670     - Glu Lys Lys Ala Leu Glu Pro Gly Ser Tyr Th - #r Ile Val Cys Ser Thr     #       685     - Phe Ala Pro Asp Gln Leu Gly Arg Phe Thr Le - #u Trp Val Ser Ser Leu     #   700     - Val Pro Cys Lys Thr Ser Pro Leu Pro Pro Gl - #u Ala Ala Gly Arg Arg     705                 7 - #10                 7 - #15                 7 -     #20     - Thr Val Ile Ser Asp Ile Gly Val Leu Pro Pr - #o Gly Arg Asp Arg Met     #               735     - Leu Ala Ser Leu Gln Val Pro Arg Leu Thr Ar - #g Ile Lys Leu Ile Thr     #           750     - Arg Ser Arg Gln Ser Ile Ile Gly Ser His Pr - #o Val Gly Pro Ser Pro     #       765     - Val Leu Met Thr Val Glu Leu Gly Gln Gly Pr - #o Tyr Lys Gln Ile Leu     #   780     - Ala Thr Ser Glu Asp Gly Thr His Ser Asp Al - #a Val Ser Gly Val Arg     785                 7 - #90                 7 - #95                 8 -     #00     - Val Glu Asp Phe Asp Leu Gln Pro Gly Leu Gl - #u Glu Ser Gly Gly Ile     #               815     - Trp Ile Val Ile Glu Arg Ile Gly Gly Pro Gl - #y Gly Gln Val Glu Asp     #           830     - His Phe Glu Val Glu Ala Leu Ala Glu Glu Ar - #g Val Glu Ile Gly Glu     #       845     - Trp Ile Leu Glu Asp Ala         850     - (2) INFORMATION FOR SEQ ID NO:18:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 842 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: None     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:     - Met Ser Arg Thr Ser Ser Ala Pro Ser Gln Ly - #s Ser Leu Ile Ser Arg     #                15     - Ala Leu Lys Ala Glu Arg Asp Val Ile Thr Al - #a Ser Ser Gln Ser Gln     #            30     - Ala Leu Asp Ala Ala Ile Asp Ala Ala Glu Hi - #s Tyr Met Lys Ala Leu     #        45     - Ala Leu Thr Ser Ser Ser Lys Asp Arg Asn Va - #l Leu Asp Ala Lys Cys     #    60     - Lys Glu Trp Leu Thr Arg Ala Glu Lys Ile Ly - #s Gly Ser Glu Asp Trp     #80     - Arg Ser Val Ala Gln Ser Arg Arg Ser Arg Le - #u Arg Thr Pro Ala Ser     #                95     - Thr Arg Lys Leu Thr Thr Arg Glu Asp Ile Il - #e Leu Leu Gln Gly Ala     #           110     - Lys Leu Asn Gly Phe Ile Phe Pro Pro Trp Ly - #s Ala Glu Pro Ser Leu     #       125     - Thr Glu Phe Glu Thr Gly Thr Asn Gly Asp Va - #l Leu Phe Thr Asp Lys     #   140     - Pro Asp Leu His Leu Ser Asn Leu Gln Arg As - #p Ile Phe Ala Gly Trp     145                 1 - #50                 1 - #55                 1 -     #60     - Lys Arg Pro His Glu Leu Leu Ser Gly Gln Va - #l Asp Asp Ala Gly Met     #               175     - Pro Leu Asn Pro Val Met Thr Val Ser Gly As - #n Thr Asp Leu Val Gln     #           190     - Asp Val Leu Thr Asp Cys Ser Val Val Ala Se - #r Leu Cys Ala Thr Thr     #       205     - Ser Arg Ser Glu Arg Gly Leu Asp Asp Thr Le - #u Leu Pro Ile Val Tyr     #   220     - Pro Cys Ile His Asn Ser Met Lys Ser Asp Il - #e Ser Pro Ser Gly Lys     225                 2 - #30                 2 - #35                 2 -     #40     - Tyr Ile Phe Arg Phe Tyr Phe Asn Gly Cys Ph - #e Arg Lys Val Val Ile     #               255     - Asp Asp Arg Leu Pro Ser Ser Lys Thr Ser Ar - #g Ser Leu Tyr Met Ile     #           270     - Asp Arg Asn His Arg Asn Phe Met Trp Pro Al - #a Leu Val Glu Lys Ala     #       285     - Tyr Leu Lys Leu Arg Gly Gly Tyr Glu Phe Pr - #o Gly Ser Asn Ser Gly     #   300     - Thr Asp Leu Trp Val Leu Thr Gly Trp Ile Pr - #o Glu Gln Val Phe Leu     305                 3 - #10                 3 - #15                 3 -     #20     - His Ser Asp Glu Val Thr Ala Asp Gln Ile Tr - #p Ser Asp Leu Phe Lys     #               335     - Ser Phe His Ser Gly Asp Val Leu Leu Thr Il - #e Gly Thr Gly Lys Leu     #           350     - Thr Glu Arg Glu Gln Lys Glu Leu Gly Leu Va - #l Ser Glu His Asp Tyr     #       365     - Ala Ile Leu Asp Met Lys Glu Leu Lys Gly Ar - #g Arg Gln Phe Leu Ile     #   380     - Lys Asn Pro Trp Ala Gly Thr Asp Ala Val Ty - #r Pro Ala Leu Phe Ala     385                 3 - #90                 3 - #95                 4 -     #00     - Asp Pro Gly Pro Phe Pro Asn Ser Pro Phe Le - #u Ser Pro Gly Thr Phe     #               415     - Trp Met Asp Cys Glu Met Val Leu Gln Asn Ph - #e Glu Asn Leu Tyr Leu     #           430     - Asn Trp Asn Pro Gly Ile Phe Ala Tyr Gln Gl - #u Asp Ile His Phe Thr     #       445     - Trp Asp Leu Ser Thr Gly Lys Gly Met Ala Gl - #y Cys Phe Val Lys Asn     #   460     - Pro Gln Phe Ser Val Tyr Thr Glu Arg Gly Gl - #y Val Val Trp Leu Leu     465                 4 - #70                 4 - #75                 4 -     #80     - Leu Gly Arg His Leu Arg Thr Ile Glu Ser Ar - #g Ala Ser Glu Glu Asp     #               495     - Glu Arg Phe Gly Phe Ile Ser Ile Tyr Val Ph - #e Lys Gly Gly Lys Arg     #           510     - Val Ala Leu Ser Asp Gly Ala Leu His Arg Gl - #y Pro Tyr Val Asp Ser     #       525     - Pro Asn Thr Leu Met Lys Leu Asp Val Pro Pr - #o Arg Ser Thr Tyr Thr     #   540     - Ala Val Val Ser Glu Glu Ser Leu Pro Arg Va - #l Ser Gln Asn Phe Thr     545                 5 - #50                 5 - #55                 5 -     #60     - Ile Ser Ala Phe Ser Asp Ser Pro Val Arg Il - #e Ser His Ala Pro Asn     #               575     - Lys Tyr Ile Cys Val Thr Lys Val Gln Gly Se - #r Trp Thr Pro Thr Thr     #           590     - Ala Gly Gly Asn Ala Glu Ser Ala Arg Tyr Se - #r Leu Asn Pro Gln Phe     #       605     - Ser Ile Val Leu Ser Asp Pro Thr Asp Ile Se - #r Ile Val Leu Glu Pro     #   620     - Ser Asp Gln Glu Leu Ala Thr His Val Lys Le - #u Phe Trp Ser Gly Gly     625                 6 - #30                 6 - #35                 6 -     #40     - Lys Arg Ile Ala Arg Val Arg Ser Arg Asp Il - #e Val Ala Asp Ser Gly     #               655     - Asp Tyr Arg Arg Gly Gly Ser Leu Val Glu Ly - #s Gln Asp Leu Asp Pro     #           670     - Gly Glu Tyr Thr Ile Val Val Ser Thr Phe Al - #a Pro Asp Gln Tyr Gly     #       685     - Ser Phe Thr Leu Trp Val Ser Thr Asn Ile Th - #r Cys Glu Val Thr Gln     #   700     - Leu Pro Ser Glu Ala Ala Gly Arg Arg Ala Va - #l Leu Ser Asp Ile Gly     705                 7 - #10                 7 - #15                 7 -     #20     - Val Leu Leu Pro Gly Gln Asp Arg Met Leu Al - #a Pro Leu Thr Thr Pro     #               735     - Arg Leu Thr Arg Val Lys Leu Ile Ala Arg Se - #r Arg Glu Ser Arg Ile     #           750     - Gly Asn Arg Pro Val Gly Pro Ser Pro Leu Le - #u Met Thr Val Glu Leu     #       765     - Gly Gln Gly Pro Tyr Lys Glu Ile Leu Ala Th - #r Ser Glu Asp Gly Asp     #   780     - His Ser Asp Ser Ile Ser Gly Val Arg Val Gl - #u Asp Phe Asp Leu Gln     785                 7 - #90                 7 - #95                 8 -     #00     - Pro Gly Leu Glu Glu Arg Gly Gly Val Trp Il - #e Val Leu Glu Arg Ile     #               815     - Gly Gly Leu Ala Val Lys Trp Lys Ile Ile Se - #r Lys Trp Lys Leu Trp     #           830     - Glu Lys Arg Glu Trp Arg Leu Gly Asn Gly     #       840     - (2) INFORMATION FOR SEQ ID NO:19:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 38 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: None     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:     - Gly Ile Leu Leu Ala Gly Ser Ala Asn Ser Ly - #s Tyr Ala Phe Leu Gly     #                15     - Ser Leu Arg Ser Thr Ala Gln Leu Ile Ser Ty - #r Glu Leu Ile Leu Ser     #            30     - Ser Val Ile Leu Leu Val             35     - (2) INFORMATION FOR SEQ ID NO:20:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 371 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: None     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:     - Met Phe Tyr Ser Leu Thr Ile Ile Ser Ile Le - #u Glu Val Leu Leu Val     #                15     - Leu Val Pro Ser Leu Leu Ala Val Ala Tyr Va - #l Thr Val Ala Glu Arg     #            30     - Lys Thr Met Ala Ser Met Gln Arg Arg Leu Gl - #y Pro Asn Ala Val Gly     #        45     - Tyr Leu Gly Leu Leu Gln Ala Phe Ala Asp Al - #a Leu Lys Leu Leu Leu     #    60     - Lys Glu Tyr Val Ala Leu Thr Gln Ala Asn Me - #t Thr Leu Phe Phe Leu     #80     - Gly Pro Val Ile Thr Leu Ile Phe Ser Leu Le - #u Gly Tyr Ala Val Ile     #                95     - Pro Tyr Gly Pro Ser Leu Val Ile Gln Asp Va - #l Asn Leu Gly Ile Leu     #           110     - Tyr Met Leu Ala Val Ser Ser Leu Ala Thr Ty - #r Gly Ile Leu Leu Ala     #       125     - Gly Trp Ser Ala Asn Ser Lys Tyr Ala Phe Le - #u Gly Ser Leu Arg Ser     #   140     - Ala Ala Gln Leu Ile Ser Tyr Glu Leu Val Le - #u Ser Ser Ala Ile Leu     145                 1 - #50                 1 - #55                 1 -     #60     - Leu Val Ile Met Leu Thr Gly Ser Phe Asn Le - #u Gly Val Asn Thr Glu     #               175     - Ser Gln Arg Ala Val Leu Phe Val Leu Pro Le - #u Leu Pro Ile Phe Ile     #           190     - Ile Phe Phe Ile Gly Ser Ile Ala Glu Thr As - #n Arg Ala Pro Phe Asp     #       205     - Leu Ala Glu Ala Glu Ser Glu Leu Val Ser Gl - #y Phe Met Thr Glu His     #   220     - Ala Ala Val Val Phe Val Phe Phe Phe Leu Al - #a Glu Tyr Gly Ser Ile     225                 2 - #30                 2 - #35                 2 -     #40     - Val Leu Met Cys Ile Leu Thr Ser Ile Leu Ph - #e Leu Gly Gly Tyr Leu     #               255     - Phe Ile Asn Leu Lys Asp Val Phe Asn Ile Le - #u Asp Phe Val Tyr Ser     #           270     - Asn Leu Phe Ile Phe Glu Ile Asn Trp Met Va - #l Ser Glu Arg Ser Tyr     #       285     - Thr Glu Asp Phe Phe Asn Asn Tyr Lys Ser Il - #e Leu Glu Gly Trp Leu     #   300     - Tyr Gly Trp Ile Ile Gly Leu Lys Ser Ser Il - #e Met Ile Phe Ile Phe     305                 3 - #10                 3 - #15                 3 -     #20     - Ile Leu Gly Arg Ala Ser Phe Pro Arg Ile Ar - #g Tyr Asp Gln Leu Met     #               335     - Gly Phe Cys Trp Thr Val Leu Leu Pro Ile Il - #e Phe Ala Leu Ile Ile     #           350     - Leu Val Pro Cys Ile Leu Glu Ser Phe Tyr Il - #e Leu Pro Trp Asn Leu     #       365     - Asn Leu Phe         370     - (2) INFORMATION FOR SEQ ID NO:21:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 36 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:     #       36         CTTC GTCACCTTAT TAGCCC     - (2) INFORMATION FOR SEQ ID NO:22:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 40 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:     #    40            TCGA GATCGTGTGT CGCGAGTACG     - (2) INFORMATION FOR SEQ ID NO:23:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 41 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:     #   41             CGCG CGAACAGACA TCACAGGAAC C     - (2) INFORMATION FOR SEQ ID NO:24:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 42 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:     #  42              GAAT TCACTTCATT CCCACTGCGT GG     - (2) INFORMATION FOR SEQ ID NO:25:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 6800 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:     - TATTCTGCTA GTAGTTAGAT CTACTGAGGG GGTATAACTC TTCGGTAGCC GG - #TGCGTATG       60     - CAGTTGTATT GTGACTGATA GATGAGAAGA AGGGCGTTTA CTGGTTGCAA GA - #GAATGATG      120     - TTATGCTTTG CATATAAGAG ATGGAATTAT TACTGATATG TGTCTAAAAT TA - #CAATGATT      180     - TGTTTTACTG AAACTGAGTA TAATGCCAGT TTGGATGTAG CCGCGTAGGT GA - #TACTTAAT      240     - CCGGACCAAA TTATGAGACC TCGATAGTAC AATGCTATTG GACTTGTAAG AT - #AATAGTCT      300     - AGTTCTTCAT AGACAAACCT GAAAAAGGAA GGACTCGAAG TCATTTCGAC AA - #GCCATAGT      360     - ATGATTAGGC ATCTAGGAGC TGAACGGAAA CGTCGTATGC GAGAGGGCCA GA - #ACCAAAAT      420     - ACGACAGTAG GAGGATTCCT ACCCCTTGGT ATATATGGAA ACCGGTTTTA AA - #GACTGGGC      480     - GTCCTCGTAC CTCACAGTCG AGCATGCCAG ACGTTGGCGC ACACATTCCC CA - #GATCGGCT      540     - GGAGCCTATC GCAATGCAAC CGTACCCACC CGGCGTNTAC TATATCTGAG CA - #TTCCTGCT      600     - GCAAGTCGTG GAGGTTGTTC CAGCCAATGA GGTCCGTTGA GCTTCTTTTT CC - #GATATGGT      660     - GATAGCTGAC AGTGTACTAG TGTCGACTGC ACAGGTGCCT CGCGGAAATG TG - #AGATCGGA      720     - GCATGTCTTG CTTCAGCGAC CGAAGTATGG ACGAATGTCC GCCATCTTGA AG - #AATGGTGA      780     - CTACCGGAGG ATTGTCTCCA GCTCAGGACT ATGTATATGC CACGATGCCA CG - #GCTTTGTG      840     - TGATAGAGTT CAAGAGCCGA GATTTACAAC GGTGATTACT ATCGCAGATA TC - #AGCACAGT      900     - ACCGGCAAGA TGGTCAACTG GAGCAACATA CTACCCACAT TAACCGCCTT CA - #TCGCTTTT      960     - ATCCTGGGCA TGCTATGCCT CTTTGCAGGA ACGAAAACAA ACCTTTTATT AG - #ATACAGAT     1020     - GTCTTCACGG TGTGTGAGCC TCCGTCACAC TTGCTTGTAC GAGCGGGGAC TA - #ACGACAGG     1080     - CGAAGATATA TACGACAAGT ATAAGCAATG GCACGGGGAT GCGGGACTTC TA - #CTCGATAT     1140     - ATGTCATGTC TTACTGTGAG GGATTCCTGC ATGCAGAAAA TCGAAACCTA AC - #CGGATGCT     1200     - CACACCCGTC ACTACTGTTC TCCTTCAATG CGACAGAAGC GTTAACGAAA GA - #TGCTGGCA     1260     - ACAACACCTC GTTATCCAGC CTGGGATGGC CGAGTTCCAT CACCGATGAT CT - #ACGCACGT     1320     - TCGGTGCTAC CAGCCAGAGT ATGGGTGTCT TCTACTGTAT TGGGATAGGA TT - #AGCGGGAC     1380     - TGGCGGTTTT GGAACGATTG TGGTTCGTGA TCGCGAAAGG GCCGAGACAG AC - #GGTTGTAG     1440     - AAGTTTCTTC TCTTATGGTA GGCTCCATTG AACGTTCTAG ATCCCGTCCG GT - #ACTAAGAA     1500     - GGCCCACAGC TCAGTTTCAC TATGCTCAGC ATACCGTCTA TCATCGCAAC GG - #TCGTTGCC     1560     - TTACAATTTG TGAGCCTCAT CAATCGTCAC GGAGAGGAGT CTGGTGTGAC AG - #CGAGATAT     1620     - GGACATCAAT TTTTAGGAAT GACGTGGGCA GCCGTCGGGT TGTTGCTGGT CG - #GAAGCACT     1680     - GTCAGTTTAC TGACGGTATT GGTGGACCGC AACCGATCGG CAGACCAGTA TG - #AACCGGTG     1740     - GCAGAACCGA AGACGGTGGC CGAGGATTCG GACTCGGTAG CGTCGAACCA GA - #AGGGGGAC     1800     - TAAGAAAGGA ACAGAATGGA GAGGAATAAT CATAAGAGAA AAAAAGGGGG AA - #ATTAACCA     1860     - AAGCAGGAAA AGTAGGGAAA AAAAAAAGAA GAAGACCGGA GAAAGCCAAG GA - #AAGGGAAC     1920     - GAATCGGGAG GAGTGTTTCT TGTTTGCAAA TACGTTGGAT TGGAGCCCAA TA - #TTGAATAT     1980     - ACTCCGTACT GTAGTCGAAA GAAAGGACAA GAGCCCCACC AAACCTCGAC CG - #TTCCATAG     2040     - CAGAAATTCC ATGACTATCT GTTAATATTT TCTGATCGAT GATGTTGTAA CA - #GTGGTTGA     2100     - AGTGGACCTT ATTTTTCGCA ATACAATAGC AGCATGGCTT GTGGGAAGGG GT - #CTTGAGAT     2160     - CATAGTCATA GTGTAAGAAA AAATTGGGTC TCCCGCGGAT GGCGACGTCA CG - #AGTGGACC     2220     - CGGGAAAAGG TCTCGCCAAA TGAGGCAACC CGGTTCCCTC CGTTCCAACC GC - #TATGCCTG     2280     - CGATTCTATC AATGATAGTG GGTCACTCGA TGGATTCAAG GGATGTCAAA TG - #ATGACGTT     2340     - AAGTTGACTA CGACTGTTGC TGTCATGCAC TGGAAATTTA TCGGGAGGAA TT - #GACCAGTC     2400     - TTATTCGCGG AGGAGGGAGT GAAGAGACTA GGAGTCGTTC AGTTCAGTCT TG - #CAGGAAAT     2460     - CCACTCGGGG ATGAGCAGGG GTTAACCTCA TGGACGACTG AACTACTCCG TA - #CAGGACGG     2520     - AGTACAGAAT GTCGAATTCA CCCGTTCGGC TAAAGATGAG TCCGTTTTAT GG - #TCACCTAG     2580     - CGGAGTGCAA CCCCGACTGG ATAACTGTTA GGAAAAGAGA GAATTAGGAT CG - #GGTGACAA     2640     - TTGGGTCTCT AAGTTCCTGC GTAACGTGAT ACCGAATCAA ATCCATGACC CT - #ATCGCCAG     2700     - GACGATCACG GAATGGTCCG GATCAGAAAT TCTGAGGTTC TGGTTGGATT GA - #TGACCTGA     2760     - ACTAATACCC AATATCATGA CGAAAACCAA TCCCCTCATT TCCTGTTTTT GC - #ACGGGAAT     2820     - AGCCACAATT TCCCCCCCCC CCCCCCCCCC CCAGAAAACC GAAATGAAGT CT - #GAGCCCTC     2880     - CGGAAGACTG CGTTCCTAGC CCCCATGTGT TTAGTTGATA CTATTCCCAT AG - #GCACGTTT     2940     - CCCCCCTTCC CCACTTGAGT TCCACCAGTC GTGAATGAGG AGGTTCCATT CT - #CGCCCAGA     3000     - GTTTGGTTTC TTTGCCGTTC CATACAAGGC GTCACTGCAT CATTCCTTCC CT - #TTTCATTC     3060     - ACCCCTCTCT TGTCCACCAT CGTGAAATGT TTCTGTAGTA CATTTAATAA AT - #ACCCCTCG     3120     - TTACCCCTCT TTCTGTTTCC CAAGAAATCA ACATCATCAT CAACAACAAC AA - #CAACAATC     3180     - ACCCTCCCAC TTCACAGGTT CTCTTTCTGA TACCCATCCT TCTGTCTCTC AT - #CTACTACC     3240     - ACTACTTTCA TATACTCTCT TCTATCCTAC TTCATCACCA TCACAACCTT CT - #TCCCCATT     3300     - CTTGTTTCAA CCCAACATCA ATATATTACC GTTGTCAACC ATCCATCATG GG - #TAAAAAGG     3360     - CTATCCAGTT TGGCGGTGGA AACATTGGCC GTGGCTTTGT GGCTGAGTTT CT - #CCACGCTG     3420     - CCGGCTATGA AGTCGTCTTC ATTGATGTCA TGGATAGCGT CATCAACTCT TT - #GCAACAGA     3480     - CCCCGTCGTA CGACGTCACG GAGGTCAGCG AAGAGGGTGA AAGCACCAAG AC - #CATCACCA     3540     - ACTATCGCGC CATCAACTCC AAGACGCATG AGGCCGACGT CGTTCAGGAG AT - #CGCATCGG     3600     - CAGATGTGGT TACCTGTGCT GTCGGTCCCA ACATCCTTAA GTTCATCGCG CC - #AGTCATTG     3660     - CCAAAGGTAT TGATGCGCGC ACCGAAGAGA GACCCGTGGC TGTGATCGCC TG - #TGAGAACG     3720     - CTATCGGCGC TACAGATACC TTGCACGGCT ACATCAAGCA GCACACCAAC CC - #TGACCGTC     3780     - TGGAGACCCT CTCTGAGCGT GCCCGTTTTG CCAACTCGGC TATCGACCGC AT - #CGTCCCCA     3840     - ACCAGCCCCC GAACAGTGGT CTCAATGTTC GCATCGAGAA GTTCTACGAG TG - #GGCCGTGG     3900     - AGAAGACTCC ATTTGGCGAA TGGGGTCACC CCGACATCCC TGCCATCCAC TG - #GGTGGACC     3960     - ACCTCGAACC TTACATCGAA CGCAAGCTCT TCACCGTCAA CACTGGCCAT GC - #TACCACCG     4020     - CCTACTATGC TCACAAGCGT GGCAAGAAGA TGATCGCCGA GGCCCTCGAA GA - #CCCAGAGA     4080     - TCCGCGAGAC TGTGCACAAG GTGCTCGAGG AGACTGCTTC CCTCATTGTA TC - #CAAGCATG     4140     - AGATCTCGGA GCAGGAGCAG AAGGAATACG TTGACAAGAT TGTCAGCCGT AT - #CTCCAACC     4200     - CCTATCTCGA GGACAACGTT GAGCGTGTGG GACGTGCTCC TCTCCGCAAA CT - #GTCTCGCA     4260     - AGGAACGGTT CATTGGACCT GCTTCGCAGC TCGCAGAGCG CGGCCAGAAG TT - #CGATGCTC     4320     - TCCTGGGCGC CATCGAGATG GCTCTTCGCT TCCAGAACGT CCCAGGCGAC GA - #GGAGAGTT     4380     - CCGAGCTTGC TCGCATTTTG AAGGAGAACT CGGCCGAGGA TGCCACCTCG CA - #GCTCACCG     4440     - GATTGGAGAA AGACCACCCA CTCTACTCTC ATGTGGTTGA GCGTGTGTCC AC - #GGTCCAGC     4500     - AAGGCTCCAA ATCAGTGCTG TGATTCTCGA TCGTTTTCCA CACCACCACA CT - #CCTTTTTA     4560     - TCACCAGAAA ACGAAGGGTT CCGAGTCCAT CACCAATATG GATCGCCCGA GG - #GATATTGG     4620     - ATCTGATATC AAACTGTTCT GTCCGCTGGC CGGGCATGAA CTGCATGGGA TA - #CGGCGAAC     4680     - ATATGAAATA ACCCCCAATT CCCATAAGTA TTACATATTA TGGAACCACA GC - #CGGTGTCT     4740     - GTAAATGTCG GTTCAACTCG AAGATGGCCG ATGCAATCGG CCCGTAGGGT AT - #ATGGTCTG     4800     - GCGCCACCTC GGCCGCCGGC TTCCCCCTTT TTATAGATGT GGCGAATAAA AC - #ACCGGATG     4860     - TTTTGTGTGT CAGGGGAATG GTGGCAGTGG TGTTATGAGT CATTGTGAAG TG - #AGTAGTGA     4920     - GTAGATTTGG TGGGGATTTT CATAGATGGT GGTTTGAAGG TCTTGGGTTT CT - #GGGGTTTA     4980     - TCCGCGTATA TTCTGCTAGT AGTTAGATCT ACTGAGGGGG TATAACTCTT CG - #GTAGCCGG     5040     - TGCGTATGCA GTTGTATTGT GACTGATAGA TGAGAAGAAG GGCGTTTACT GG - #TTGCAAGA     5100     - GAATGATGTT ATGCTTTGCA TATAAGAGAT GGAATTATTA CTGATATGTG TC - #TAAAATTA     5160     - CAATGATTTG TTTTACTGAA ACTGAGTATA ATGCCAGTTT GGATGTAGCC GC - #GTAGGTGA     5220     - TACTTAATCC GGACCAAATT ATGAGACCTC GATAGTACAA TGCTATTGGA CT - #TGTAAGAT     5280     - AATAGTCTAG TTCTTCATAG ACAAACCTGA AAAAGGAAGG ACTCGAAGTC AT - #TTCGACAA     5340     - GCCATAGTAT GATTAGGCAT CTAGGAGCTG AACGGAAACG TCGTATGCGA GA - #GGGCCAGA     5400     - ACCAAAATAC GACAGTAGGA GGATTCCTAC CCCTTGGTAT ATATGGAAAC CG - #GTTTTAAA     5460     - GACTGGGCGT CCTCGTACCT CACAGTCGAG CATGCCAGAC GTTGGCGCAC AC - #ATTCCCCA     5520     - GATCGGCTGG AGCCTATCGC AATGCAACCG TACCCACCCG GCGTCTACTA TA - #TCTGAGCA     5580     - TTCCTGCTGC AAGTCGTGGA GGTTGTTCCA GCCAATGAGG TCCGTTGAGC TT - #CTTTTTCC     5640     - GATATGGTGA TAGCTGACAG TGTACTAGTG TCGACTGCAC AGGTGCCTCG CG - #GAAATGTG     5700     - AGATCGGAGC ATGTCTTGCT TCAGCGACCG AAGTATGGAC GAATGTCCGC CA - #TCTTGAAG     5760     - AATGGTGACT ACCGGAGGAT TGTCTCCAGC TCAGGACTAT GTATATGCCA CG - #ATGCCACG     5820     - GCTTTGTGTG ATAGAGTTCA AGAGCCGAGA TTTACAACGG TGATTACTAT CG - #CAGATATC     5880     - AGCACAGTAC CGGCAAGATG GTCAACTGGA GCAACATACT ACCCACATTA AC - #CGCCTTCA     5940     - TCGCTTTTAT CCTGGGCATG CTATGCCTCT TTGCAGGAAC GAAAACAAAC CT - #TTTATTAG     6000     - ATACAGATGT CTTCACGGTG TGTGAGCCTC CGTCACACTT GCTTGTACGA GC - #GGGGACTA     6060     - ACGACAGGCG AAGATATATA CGACAAGTAT AAGCAATGGC ACGGGGATGC GG - #GACTTCTA     6120     - CTCGATATAT GTCATGTCTT ACTGTGAGGG ATTCCTGCAT GCAGAAAATC GA - #AACCTAAC     6180     - CGGATGCTCA CACCCGTCAC TACTGTTCTC CTTCAATGCG ACAGAAGCGT TA - #ACGAAAGA     6240     - TGCTGGCAAC AACACCTCGT TATCCAGCCT GGGATGGCCG AGTTCCATCA CC - #GATGATCT     6300     - ACGCACGTTC GGTGCTACCA GCCAGAGTAT GGGTGTCTTC TACTGTATTG GG - #ATAGGATT     6360     - AGCGGGACTG GCGGTTTTGG AACGATTGTG GTTCGTGATC GCGAAAGGGC CG - #AGACAGAC     6420     - GGTTGTAGAA GTTTCTTCTC TTATGGTAGG CTCCATTGAA CGTTCTAGAT CC - #CGTCCGGT     6480     - ACTAAGAAGG CCCACAGCTC AGTTTCACTA TGCTCAGCAT ACCGTCTATC AT - #CGCAACGG     6540     - TCGTTGCCTT ACAATTTGTG AGCCTCATCA ATCGTCACGG AGAGGAGTCT GG - #TGTGACAG     6600     - CGAGATATGG ACATCAATTT TTAGGAATGA CGTGGGCAGC CGTCGGGTTG TT - #GCTGGTCG     6660     - GAAGCACTGT CAGTTTACTG ACGGTATTGG TGGACCGCAA CCGATCGGCA GA - #CCAGTATG     6720     - AACCGGTGGC AGAACCGAAG ACGGTGGCCG AGGATTCGGA CTCGGTAGCG TC - #GAACCAGA     6780     #                 680 - #0     - (2) INFORMATION FOR SEQ ID NO:26:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 391 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: None     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:     - Met Gly Lys Lys Ala Ile Gln Phe Gly Gly Gl - #y Asn Ile Gly Arg Gly     #                15     - Phe Val Ala Glu Phe Leu His Ala Ala Gly Ty - #r Glu Val Val Phe Ile     #            30     - Asp Val Met Asp Ser Val Ile Asn Ser Leu Gl - #n Gln Thr Pro Ser Tyr     #        45     - Asp Val Thr Glu Val Ser Glu Glu Gly Glu Se - #r Thr Lys Thr Ile Thr     #    60     - Asn Tyr Arg Ala Ile Asn Ser Lys Thr His Gl - #u Ala Asp Val Val Gln     #80     - Glu Ile Ala Ser Ala Asp Val Val Thr Cys Al - #a Val Gly Pro Asn Ile     #                95     - Leu Lys Phe Ile Ala Pro Val Ile Ala Lys Gl - #y Ile Asp Ala Arg Thr     #           110     - Glu Glu Arg Pro Val Ala Val Ile Ala Cys Gl - #u Asn Ala Ile Gly Ala     #       125     - Thr Asp Thr Leu His Gly Tyr Ile Lys Gln Hi - #s Thr Asn Pro Asp Arg     #   140     - Leu Glu Thr Leu Ser Glu Arg Ala Arg Phe Al - #a Asn Ser Ala Ile Asp     145                 1 - #50                 1 - #55                 1 -     #60     - Arg Ile Val Pro Asn Gln Pro Pro Asn Ser Gl - #y Leu Asn Val Arg Ile     #               175     - Glu Lys Phe Tyr Glu Trp Ala Val Glu Lys Th - #r Pro Phe Gly Glu Trp     #           190     - Gly His Pro Asp Ile Pro Ala Ile His Trp Va - #l Asp His Leu Glu Pro     #       205     - Tyr Ile Glu Arg Lys Leu Phe Thr Val Asn Th - #r Gly His Ala Thr Thr     #   220     - Ala Tyr Tyr Ala His Lys Arg Gly Lys Lys Me - #t Ile Ala Glu Ala Leu     225                 2 - #30                 2 - #35                 2 -     #40     - Glu Asp Pro Glu Ile Arg Glu Thr Val His Ly - #s Val Leu Glu Glu Thr     #               255     - Ala Ser Leu Ile Val Ser Lys His Glu Ile Se - #r Glu Gln Glu Gln Lys     #           270     - Glu Tyr Val Asp Lys Ile Val Ser Arg Ile Se - #r Asn Pro Tyr Leu Glu     #       285     - Asp Asn Val Glu Arg Val Gly Arg Ala Pro Le - #u Arg Lys Leu Ser Arg     #   300     - Lys Glu Arg Phe Ile Gly Pro Ala Ser Gln Le - #u Ala Glu Arg Gly Gln     305                 3 - #10                 3 - #15                 3 -     #20     - Lys Phe Asp Ala Leu Leu Gly Ala Ile Glu Me - #t Ala Leu Arg Phe Gln     #               335     - Asn Val Pro Gly Asp Glu Glu Ser Ser Glu Le - #u Ala Arg Ile Leu Lys     #           350     - Glu Asn Ser Ala Glu Asp Ala Thr Ser Gln Le - #u Thr Gly Leu Glu Lys     #       365     - Asp His Pro Leu Tyr Ser His Val Val Glu Ar - #g Val Ser Thr Val Gln     #   380     - Gln Gly Ser Lys Ser Val Leu     385                 3 - #90     - (2) INFORMATION FOR SEQ ID NO:27:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 195 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: None     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:     - Met Val Asp Gln Ala Gln Asp Thr Leu Arg Pr - #o Asn Asn Arg Leu Ser     #                15     - Asp Met Gln Ala Thr Met Glu Gln Thr Gln Al - #a Phe Glu Asn Arg Val     #            30     - Leu Glu Arg Leu Asn Ala Gly Lys Thr Val Ar - #g Ser Phe Leu Ile Thr     #        45     - Ala Val Glu Leu Leu Thr Glu Ala Val Asn Le - #u Leu Val Leu Gln Val     #    60     - Phe Arg Lys Asp Asp Tyr Ala Val Lys Tyr Al - #a Val Glu Pro Leu Leu     #80     - Asp Gly Asp Gly Pro Leu Gly Asp Leu Ser Va - #l Arg Leu Lys Leu Ile     #                95     - Tyr Gly Leu Gly Val Ile Asn Arg Gln Glu Ty - #r Glu Asp Ala Glu Leu     #           110     - Leu Met Ala Leu Arg Glu Glu Leu Asn His As - #p Gly Asn Glu Tyr Ala     #       125     - Phe Thr Asp Asp Glu Ile Leu Gly Pro Phe Gl - #y Glu Leu His Cys Val     #   140     - Ala Ala Leu Pro Pro Pro Pro Gln Phe Glu Pr - #o Ala Asp Ser Ser Leu     145                 1 - #50                 1 - #55                 1 -     #60     - Tyr Ala Met Gln Ile Gln Arg Tyr Gln Gln Al - #a Val Arg Ser Thr Met     #               175     - Val Leu Ser Leu Thr Glu Leu Ile Ser Lys Il - #e Ser Leu Lys Lys Ala     #           190     - Phe Gln Lys             195     - (2) INFORMATION FOR SEQ ID NO:28:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 366 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: None     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:     - Met Ile Ala Leu His Phe Gly Ala Gly Asn Il - #e Gly Arg Gly Phe Ile     #                15     - Gly Ala Leu Leu His His Ser Gly Tyr Asp Va - #l Val Phe Ala Asp Val     #            30     - Asn Glu Thr Met Val Ser Leu Leu Asn Glu Ly - #s Lys Glu Tyr Thr Val     #        45     - Glu Leu Ala Glu Glu Gly Arg Ser Ser Glu Il - #e Ile Gly Pro Val Ser     #    60     - Ala Ile Asn Ser Gly Ser Gln Thr Glu Glu Le - #u Tyr Arg Leu Met Asn     #80     - Glu Ala Ala Leu Ile Thr Thr Ala Val Gly Pr - #o Asn Val Leu Lys Leu     #                95     - Ile Ala Pro Ser Ile Ala Glu Gly Leu Arg Ar - #g Arg Asn Thr Ala Asn     #           110     - Thr Leu Asn Ile Ile Ala Cys Glu Asn Met Il - #e Gly Gly Ser Ser Phe     #       125     - Leu Lys Lys Glu Ile Tyr Ser His Leu Thr Gl - #u Ala Glu Gln Lys Ser     #   140     - Val Ser Glu Thr Leu Gly Phe Pro Asn Ser Al - #a Val Asp Arg Ile Val     145                 1 - #50                 1 - #55                 1 -     #60     - Pro Ile Gln His His Glu Asp Pro Leu Lys Va - #l Ser Val Glu Pro Phe     #               175     - Phe Glu Trp Val Ile Asp Glu Ser Gly Phe Ly - #s Gly Lys Thr Pro Val     #           190     - Ile Asn Gly Ala Leu Phe Val Asp Asp Leu Th - #r Pro Tyr Ile Glu Arg     #       205     - Lys Leu Phe Thr Val Asn Thr Gly His Ala Va - #l Thr Ala Tyr Val Gly     #   220     - Tyr Gln Arg Gly Leu Lys Thr Val Lys Glu Al - #a Ile Asp His Pro Glu     225                 2 - #30                 2 - #35                 2 -     #40     - Ile Arg Arg Val Val His Ser Ala Leu Leu Gl - #u Thr Gly Asp Tyr Leu     #               255     - Val Lys Ser Tyr Gly Phe Lys Gln Thr Glu Hi - #s Glu Gln Tyr Ile Lys     #           270     - Asn Gln Arg Ser Leu Leu Lys Ser Phe His Ph - #e Gly Arg Cys Asp Pro     #       285     - Arg Ser Glu Val Thr Ser Gln Lys Thr Gly Ar - #g Lys Cys Arg Leu Val     #   300     - Gly Pro Ala Lys Lys Ile Lys Glu Pro Asn Al - #a Leu Ala Glu Gly Ile     305                 3 - #10                 3 - #15                 3 -     #20     - Ala Ala Ala Leu Arg Phe Asp Phe Thr Gly As - #p Pro Glu Ala Val Glu     #               335     - Leu Gln Ala Leu Ile Glu Glu Lys Asp Thr Al - #a Ala Tyr Phe Lys Arg     #           350     - Cys Ala Ala Phe Ser Pro Met Asn Arg Cys Th - #r Pro Ser Phe     #       365     - (2) INFORMATION FOR SEQ ID NO:29:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 3300 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:     - GGATTGGCTG ACTGCGTGTT GTTACTCCAG GGATTGCGAC GAGCACTTCC TA - #AGCCACCG       60     - TGCGACGGAG GCGACTCTGA GCGTGACGAT GCCCTAGCGA ACCCGCGGAA GC - #CCATCGTG      120     - GGGCGAAAAT CTTCTTCGTC GGAGCTCATG TCGGCGATTT TACGTTTTTG GC - #CGGCGGAT      180     - GGTGAGGCCG GGGGAGAATC CGAATCCATG ACCGATCGCA GATGTCAGGA TA - #AGGTGTAA      240     - GTAGTAACTC AGAGTCGTCG GAGAGGTTCG AAAGGCAATG AAACGGTCAA AC - #GACACGTT      300     - TGAGAGCCAC GAAGGAGCTG TGGGTTGAGA TACGCACGAT AACGAGAAAG GA - #AAGTTGAT      360     - TATCGGACAT TTCGGCGCGG GGAAAATTCA AGTCCGAGGG GCCGAGCAAC AA - #TGACGTTC      420     - GTTGCATCGA ATCTCCCTTC CGGTTATTTT TCCCTCTTCT TCTCCTCTTC TT - #CTTTTCTT      480     - CTTTACCCTC TCCTCTCTTT GGCATTTCGT CACTACTTTG TAACGTAACT CA - #ATTCTATT      540     - GATACATAAA AATCACATAT CAACTATGGC TGCCTCTCTT ATCCGTACCT CT - #GCCCGTAC      600     - CGCTCTTCGC GCTGGAGCTT CGGCTACTCC TAAAGCTGCG GGTGTTGCGG GT - #TTGACCTT      660     - TGCCCGTGGC AAGGCCACTC TGCCTGACCT GGCTTGTATG GCTCTCCCCT TC - #CCTTGATG      720     - TCGTCAATTT GCCCCTCTGT TGTGTTATCT TCCGTTTTGT CATCTTTCTC GG - #CTATTTTG      780     - GCAGTGCGAA TGAGTAGATG GGTTACGCTT GTCGCTCATG ACGCCCCGGA AG - #CACGTAAT      840     - GCAATGGTTG GTTGACTGAA TAACAGATGA CTATGGCGCC CTTGAGCCCT CT - #ATCTCCGG      900     - AAAGATCATG GAGCTTCACC ACAAGAACCA CCACCAGACC TATGTCAACA GC - #TACAACAC      960     - CGCCATCGAA CAGCTCCAGG AGGCCGTCGC CAAGGAGGAC ATCACCACTC AG - #ATCAACCT     1020     - CAAGCCCCTG ATCAACTTCC ACGGTGGTGG CCACATCAAC CACACTCTTT TC - #TGGGAGAA     1080     - CCTTGCCCCT AAGAGCCAGG GCGGTGGTGA GCCCCCATCT GGAGCTTTGG CC - #AAGGCCAT     1140     - CGACGAAAGC TTCGGCAGCT TGGGAGAGTT CCAGAGCAAG ATGAACGCCG CC - #CTCGCTGG     1200     - TATTCAGGGA AGCGGATGGG CTTGGCTCGT CAAGGACAAG CAGACCGGAA AC - #ATCGGCAT     1260     - CAAGACCTAT GCCGTAAGTT CCTCCTTGTG AGCGCCTAAG GATACAGGTA GC - #TAACTCCC     1320     - GACCAGAACC AGGACCCTGT CGTTGGTCAG TTCCAGCCTC TTCTCGGTAT TG - #ATGCTTGG     1380     - GAGCACGCCT ACTAGTAAGT TTTCTTGGAC TAGATATCTA CCAAGCAATA AC - #TAATGCCG     1440     - TGTTAGCCTT CAATACCAGA ACCGCAAGGC TGAGTACTTC AGCGCCATCT GG - #GACGTCAT     1500     - CAACTGGAAG GCGGTTGAGA AGCGCTTCTC GTAAGCGTGC AAAAGTGTTG TG - #AATTGACG     1560     - CAGCTTGATG AGCGCTTTGT TTCAGTTGTG CCCAGAGTGA TACTGTGTAA TG - #TCTGATCA     1620     - AGCTGTACTT GTAGCCCTAA TGCAATTGGA TACGCCTCGT GTATATATAA AC - #TCATGTTC     1680     - GTTGAACGTA AATAATTTTG GGGAAGCTGC ACCAGCCACA GTGGCTGGAT CA - #CATGCTCC     1740     - CGTAGCATTC CCGCAGTTTC CGGCAAGCTT ATTTTCTTAG TTTGGGATCC GC - #TCCGCCCT     1800     - CTCCGGATCT TCTTCCCTCA TCTCACCTCT CAAGCGATCA ATTCTCTCGA AA - #TGTCTGCA     1860     - GAAAGCCCGG GAGAAAAGCG CGGTGGGTTT CGGGCGTTCT TCGCCGGCGC CC - #TCCGACCT     1920     - AAGAAATCCC GTCAGGTCCT CCGAAAGGCA TCGACACCGA ATCTAAAGGA AG - #GTCTACAA     1980     - AGCAAAGATG ACGTCCCGGC GATGCCTTCA CTGACCCCAT TGGAGGCCCA CC - #GACTCAAA     2040     - TACCGAGAAG TAAATCTTCA GAAAGACACA CAGCTAGGCG AAACCCACGA TC - #ATACCGCA     2100     - ATGCTGCATT CAATCGGTGT TGGAGAGCTC GATCCGTCCG ATCCACACGC GC - #AACTACAC     2160     - GAATTCGACA ATAGACCCCC AGGCGAGCCT ATGATTGCGA GCTTAACATC GG - #ACCTCTGG     2220     - GCCAAGGTCA CCGAGTATCT CAATCCCGCC GAAAGAGCCA GTCTTGCCTT CT - #CCAGCCGA     2280     - ACACTATACG CTCGTCTGGG CCGCGAGCCC TGGATAACAA TAAACCTCCC AG - #AAAACCAC     2340     - GACTACAAAG CGGACTTTCT CATCTCCCAA GATAGACTAC TCCCTCACCA TC - #TCCTCTGT     2400     - TTCCCCTGCG GCAAATACCA CCGCCGCACA CAAGAAGGCT ACGAAAAGCT CC - #AACCCGCA     2460     - GACATAATCA ACCCGCTCTT CGATTGCCCC AACGCCCGCA ACAACGCCCT CC - #CAGCACCC     2520     - CGCCACCGCA TCACCCACGG CCGAGTCCTT TACTTCACCT TCCACCAGCT AG - #TCATGCGC     2580     - GCATACCGAT TTGGACCCCG CTACGGCATC TCAGCCGACT CTCTATCCCG TC - #GCTGGCGC     2640     - CGGGACGGCT GGTCCCACCA AACCCGATAC CACATCCATC AGGGTCGACT GC - #TCATGCGA     2700     - GTCGTGAGCA CCTGCTTCGC CGAACCAGGC CTCAGCGCCA GCCAACAGCG AC - #TCCTCCTC     2760     - TACTCGCGCG ACGACTACTG GCCGTACTTC TCCGTCTGCG CGCACTGGCG GG - #ATGGCGAA     2820     - CTTATGAACG TTTGCAAATG CGCCCTCGGC CACATCCCCG TCCCCCGCAC CA - #CGAACGGC     2880     - CTGCAGGGCC TCGAACACCG CGCAAAAGAT ATGTACCACC GTCGAGAGCA CA - #ATCCCAAC     2940     - GCCCTCGCGT CGCTCTGCGG TAAGTGTCGA CCTATGCGTC GCTGCCCCGA GT - #GTCCCTCC     3000     - GAGTATCTGG TCGAGGTCAA GCTCACCGAG GACCGGAGTG GTTCGCATCG CA - #ACTTATTC     3060     - CGGCATGCGA TTGTGGTGAC ACGGTGGAGT GATTTGGGGG ATGGGCGGTC GC - #CGCGGCTA     3120     - TCGAAGGAGT GGGCGGCGAT TAATGGGGAC GAGGCGGGTG AGGGGTATGA TT - #CTTTTGAG     3180     - AAAATAGGGA AGAGGGCTAT TTCGGGGATT TTTGAGTCGG CTATTACCGA TG - #ATACTTTG     3240     - CCTGGGCAGA GGATTCTTTC AATGAATCCT AAGGGAAAGA AGTTGGGTGA GG - #CTGGGAAT     3300     - (2) INFORMATION FOR SEQ ID NO:30:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 230 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: None     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:     - Met Ala Ala Ser Leu Ile Arg Thr Ser Ala Ar - #g Thr Ala Leu Arg Ala     #                15     - Gly Ala Ser Ala Thr Pro Lys Ala Ala Gly Va - #l Ala Gly Leu Thr Phe     #            30     - Ala Arg Gly Lys Ala Thr Leu Pro Asp Leu Al - #a Tyr Asp Tyr Gly Ala     #        45     - Leu Glu Pro Ser Ile Ser Gly Lys Ile Met Gl - #u Leu His His Lys Asn     #    60     - His His Gln Thr Tyr Val Asn Ser Tyr Asn Th - #r Ala Ile Glu Gln Leu     #80     - Gln Glu Ala Val Ala Lys Glu Asp Ile Thr Th - #r Gln Ile Asn Leu Lys     #                95     - Pro Leu Ile Asn Phe His Gly Gly Gly His Il - #e Asn His Thr Leu Phe     #           110     - Trp Glu Asn Leu Ala Pro Lys Ser Gln Gly Gl - #y Gly Glu Pro Pro Ser     #       125     - Gly Ala Leu Ala Lys Ala Ile Asp Glu Ser Ph - #e Gly Ser Leu Gly Glu     #   140     - Phe Gln Ser Lys Met Asn Ala Ala Leu Ala Gl - #y Ile Gln Gly Ser Gly     145                 1 - #50                 1 - #55                 1 -     #60     - Trp Ala Trp Leu Val Lys Asp Lys Gln Thr Gl - #y Asn Ile Gly Ile Lys     #               175     - Thr Tyr Ala Asn Gln Asp Pro Val Val Gly Gl - #n Phe Gln Pro Leu Leu     #           190     - Gly Ile Asp Ala Trp Glu His Ala Tyr Tyr Le - #u Gln Tyr Gln Asn Arg     #       205     - Lys Ala Glu Tyr Phe Ser Ala Ile Trp Asp Va - #l Ile Asn Trp Lys Ala     #   220     - Val Glu Lys Arg Phe Ser     225                 2 - #30     - (2) INFORMATION FOR SEQ ID NO:31:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 233 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: None     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:     - Met Phe Ala Lys Thr Ala Ala Ala Asn Leu Th - #r Lys Lys Gly Gly Leu     #                15     - Ser Leu Leu Ser Thr Thr Ala Arg Arg Thr Ly - #s Val Thr Leu Pro Asp     #            30     - Leu Lys Trp Asp Phe Gly Ala Leu Glu Pro Ty - #r Ile Ser Gly Gln Ile     #        45     - Asn Glu Leu His Tyr Thr Lys His His Gln Th - #r Tyr Val Asn Gly Phe     #    60     - Asn Thr Ala Val Asp Gln Phe Gln Glu Leu Se - #r Asp Leu Leu Ala Lys     #80     - Glu Pro Ser Pro Ala Asn Ala Arg Lys Met Il - #e Ala Ile Gln Gln Asn     #                95     - Ile Lys Phe His Gly Gly Gly Phe Thr Asn Hi - #s Cys Leu Phe Trp Glu     #           110     - Asn Leu Ala Pro Glu Ser Gln Gly Gly Gly Gl - #u Pro Pro Thr Gly Ala     #       125     - Leu Ala Lys Ala Ile Asp Glu Gln Phe Gly Se - #r Leu Asp Glu Leu Ile     #   140     - Lys Leu Thr Asn Thr Lys Leu Ala Gly Val Gl - #n Gly Ser Gly Trp Ala     145                 1 - #50                 1 - #55                 1 -     #60     - Phe Ile Val Lys Asn Leu Ser Asn Gly Gly Ly - #s Leu Asp Val Val Gln     #               175     - Thr Tyr Asn Gln Asp Thr Val Thr Gly Pro Le - #u Val Pro Leu Val Ala     #           190     - Ile Asp Ala Trp Glu His Ala Tyr Tyr Leu Gl - #n Tyr Gln Asn Lys Lys     #       205     - Ala Asp Tyr Phe Lys Ala Ile Trp Asn Val Va - #l Asn Trp Lys Glu Ala     #   220     - Ser Arg Arg Phe Asp Ala Gly Lys Ile     225                 2 - #30     - (2) INFORMATION FOR SEQ ID NO:32:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 36 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:     #       36         GCTC ATGTCGGCGA TTTTAC     - (2) INFORMATION FOR SEQ ID NO:33:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 36 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:     #       36         AGCA AAGTATAAAT AAGGAA     - (2) INFORMATION FOR SEQ ID NO:34:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 346 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:     - ATATCGACTG TAGTAATATC TCAGGTCTCT GGGATAGCTG ATGGAATGTT AA - #GTGAATAA       60     - TATTGATTTA AAGTTCCTCT AGTTCCAAGC TCTTATGTAG CTTCATTTTC TA - #TATATATA      120     - TATTCTATTT AGTGGTGTTG CAGGCGGTGA GCCTATCGGC CAATCATAGT AA - #AAAACCCG      180     - TTAGTTGCAA TACCCTGTTA GTTGCAAGGC GAATTCCTGG CTGATATCCT TG - #CAACTAAC      240     - GGGGTTTCTC AGTACTCGAA TTGAATATAT ATTTCGCACA AAGTTATTTC GC - #AAACTTGG      300     #                346AAC CCAAGCCACA AGCTTTATTT AATTCG     - (2) INFORMATION FOR SEQ ID NO:35:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:     #  18              GG     - (2) INFORMATION FOR SEQ ID NO:36:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:     #  18              TC     - (2) INFORMATION FOR SEQ ID NO:37:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:     #  18              TT     - (2) INFORMATION FOR SEQ ID NO:38:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:     #  18              TG     - (2) INFORMATION FOR SEQ ID NO:39:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 188 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:     - GATTTAATGA CTACCTTGAT GATACTGCCA ATATAGTTAG ATAATACAAA TC - #CTGGCTGC       60     - CATATAACGC CCTCGCAAAC GACATCTTGT TCTTATTNTC CCTCAATCGA GC - #TTGCCTAT      120     - GCCCAAGCTT CGAACTATAC GAGCATTGTA AATTGATTTT GATACGGCCT GC - #CATATCAG      180     #         188     - (2) INFORMATION FOR SEQ ID NO:40:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:     #  18              TG     - (2) INFORMATION FOR SEQ ID NO:41:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:     #  18              CC     - (2) INFORMATION FOR SEQ ID NO:42:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 17 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #42:     #   17             C     - (2) INFORMATION FOR SEQ ID NO:43:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:     #  18              TG     - (2) INFORMATION FOR SEQ ID NO:44:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 27 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:     #             27   GTGG ATCAAGC     - (2) INFORMATION FOR SEQ ID NO:45:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 28 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:     #             28   GTCT GCGCTGGT     - (2) INFORMATION FOR SEQ ID NO:46:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 39 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #46:     #    39            TAAT TAATCACTCC GTACCTGAT     - (2) INFORMATION FOR SEQ ID NO:47:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 17 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:     #   17             T     - (2) INFORMATION FOR SEQ ID NO:48:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:     #  18              CA     - (2) INFORMATION FOR SEQ ID NO:49:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 21 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:     #21                TTCA G     - (2) INFORMATION FOR SEQ ID NO:50:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 1132 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:     - AACCCTGTAT GTGAGCCTGA TTCAAGACTT CGGATTTACT TTCAAGGCTT CG - #AATCACTG       60     - TTAAGGCAGA AGAAAGTAGT ACTAATGGTT ATCAATATAT AGGAGGGCAA GC - #GGGAGGTT      120     - ATCGCCAACG AAGAAGGAGG TTAGTCCACT ACTGCTTGGT GGCGGGTAAT CT - #TAAGAGCA      180     - CAAACTAACG GATACACAGA TCGTCAAATC CCTTCCGTCC TTTCATACAT TG - #ATGGTGAG      240     - GAGTACCACG GTACTCAAGC CAAGGCCCAG TTGGTCCGCA ACTCCCAGAA CA - #CTGTCGCA      300     - TACTTCAGAG ATTACCTTGG CAAGGAGTTC AAGTCGATAG ACGCCACACC AT - #GCCATAAC      360     - TCGGCGCATC CTCAGCCTCA CGAGTCTACC GTTGCTTTCT CCATTGTGGA CT - #CTACCAAC      420     - GAGACCCCCA GCACTGTCAC CGTCTCCGAG ATTGCCACCC GCCATCTCCG TC - #GTTTGAAG      480     - CAGTCCGCCT CTGACTACCT GGGCAAGGAA GTCAATGCCG CCGTCATCAC TG - #TCCCCACT      540     - GACTTCTCCG ATGCTCAGCG CGAGGCTTTG ACCGCTTCCG CTAAGGCTGC TG - #GCCTTGAG      600     - GTCCTCCAGC TCATCCATGA GCCTGTTGCC GCTGCCCTGG CTTACGATGC CA - #GGCCCGAG      660     - GCTACTGTTA CTGACAAGCT TGTTGTCGTC GCCGACCTCG GTGGTACCCG AT - #CCGACGCT      720     - GCTGTTCTCG CTTGCCGTGG TGGCATGTAC AGTATCCTCG CAACTGCTCA TG - #ACTACGAG      780     - TTGGGTGGAG CTTCGTTGGA CAAGATCATC ATCGACCATT TCGCCAAGGA GT - #TCATTAAG      840     - AAGCACAAGA CCGATCCTCG CGAGAACGCT CGTGGTCTCG CCAAGTTGAA GC - #TTGAGGGT      900     - GAGGCTGCTC GCAAGACCTT GAGCTTGGGT ACCAACGCCA GCTTGAGCAT TG - #AGATCTTC      960     - GCAGATGGCA TTGATTTCGG CTCCACTGTC AACCGTACTC GNTACGAACT TC - #TTTCCGGC     1020     - AAGACCTTCG CCCAGTTCAC CGGCTTGATC GAGCAGGTTA TCCAGAAGGC TG - #GTTTGGAT     1080     - GTTTTGGACA TTGACGAGGT TAGTCCCTTG TGATTTTTTT TTTTTTTTTC AG - #     1132     - (2) INFORMATION FOR SEQ ID NO:51:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 374 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: None     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:     - Asn Pro Val Cys Glu Pro Asp Ser Arg Leu Ar - #g Ile Tyr Phe Gln Gly     #                15     - Phe Glu Ser Leu Leu Arg Gln Lys Lys Val Va - #l Leu Met Val Ile Asn     #            30     - Ile Glu Gly Lys Arg Glu Val Ile Ala Asn Gl - #u Glu Gly Gly Ser Thr     #        45     - Thr Ala Trp Trp Arg Val Ile Leu Arg Ala Gl - #n Thr Asn Gly Tyr Thr     #    60     - Asp Arg Gln Ile Pro Ser Val Leu Ser Tyr Il - #e Asp Gly Glu Glu Tyr     #80     - His Gly Thr Gln Ala Lys Ala Gln Leu Val Ar - #g Asn Ser Gln Asn Thr     #                95     - Val Ala Tyr Phe Arg Asp Tyr Leu Gly Lys Gl - #u Phe Lys Ser Ile Asp     #           110     - Ala Thr Pro Cys His Asn Ser Ala His Pro Gl - #n Pro His Glu Ser Thr     #       125     - Val Ala Phe Ser Ile Val Asp Ser Thr Asn Gl - #u Thr Pro Ser Thr Val     #   140     - Thr Val Ser Glu Ile Ala Thr Arg His Leu Ar - #g Arg Leu Lys Gln Ser     145                 1 - #50                 1 - #55                 1 -     #60     - Ala Ser Asp Tyr Leu Gly Lys Glu Val Asn Al - #a Ala Val Ile Thr Val     #               175     - Pro Thr Asp Phe Ser Asp Ala Gln Arg Glu Al - #a Leu Thr Ala Ser Ala     #           190     - Lys Ala Ala Gly Leu Glu Val Leu Gln Leu Il - #e His Glu Pro Val Ala     #       205     - Ala Ala Leu Ala Tyr Asp Ala Arg Pro Glu Al - #a Thr Val Thr Asp Lys     #   220     - Leu Val Val Val Ala Asp Leu Gly Gly Thr Ar - #g Ser Asp Ala Ala Val     225                 2 - #30                 2 - #35                 2 -     #40     - Leu Ala Cys Arg Gly Gly Met Tyr Ser Ile Le - #u Ala Thr Ala His Asp     #               255     - Tyr Glu Leu Gly Gly Ala Ser Leu Asp Lys Il - #e Ile Ile Asp His Phe     #           270     - Ala Lys Glu Phe Ile Lys Lys His Lys Thr As - #p Pro Arg Glu Asn Ala     #       285     - Arg Gly Leu Ala Lys Leu Lys Leu Glu Gly Gl - #u Ala Ala Arg Lys Thr     #   300     - Leu Ser Leu Gly Thr Asn Ala Ser Leu Ser Il - #e Glu Ile Phe Ala Asp     305                 3 - #10                 3 - #15                 3 -     #20     - Gly Ile Asp Phe Gly Ser Thr Val Asn Arg Th - #r Arg Tyr Glu Leu Leu     #               335     - Ser Gly Lys Thr Phe Ala Gln Phe Thr Gly Le - #u Ile Glu Gln Val Ile     #           350     - Gln Lys Ala Gly Leu Asp Val Leu Asp Ile As - #p Glu Val Ser Pro Leu     #       365     - Phe Phe Phe Phe Phe Ser         370     - (2) INFORMATION FOR SEQ ID NO:52:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 339 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: None     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:     - Met Ser Lys Ala Val Gly Ile Asp Leu Gly Th - #r Thr Tyr Ser Cys Val     #                15     - Ala His Phe Ala Asn Asp Arg Val Asp Ile Il - #e Ala Asn Asp Gln Gly     #            30     - Asn Arg Thr Thr Pro Ser Phe Val Ala Phe Th - #r Asp Thr Glu Arg Leu     #        45     - Ile Gly Asp Ala Ala Lys Asn Gln Ala Ala Me - #t Asn Pro Ser Asn Thr     #    60     - Val Phe Asp Ala Lys Arg Leu Ile Gly Arg As - #n Phe Asn Asp Pro Glu     #80     - Val Gln Ala Asp Met Lys His Phe Pro Phe Ly - #s Leu Ile Asp Val Asp     #                95     - Gly Lys Pro Gln Ile Gln Val Glu Phe Lys Gl - #y Glu Thr Lys Asn Phe     #           110     - Thr Pro Glu Gln Ile Ser Ser Met Val Leu Gl - #y Lys Met Lys Glu Thr     #       125     - Ala Glu Ser Tyr Leu Gly Ala Lys Val Asn As - #p Ala Val Val Thr Val     #   140     - Pro Ala Tyr Phe Asn Asp Ser Gln Arg Gln Al - #a Thr Lys Asp Ala Gly     145                 1 - #50                 1 - #55                 1 -     #60     - Thr Ile Ala Gly Leu Asn Val Leu Arg Ile Il - #e Asn Glu Pro Thr Ala     #               175     - Ala Ala Ile Ala Tyr Gly Leu Asp Lys Lys Gl - #y Lys Glu Glu His Val     #           190     - Leu Ile Phe Asp Leu Gly Gly Gly Thr Phe As - #p Val Ser Leu Leu Phe     #       205     - Ile Glu Asp Gly Ile Phe Glu Val Lys Ala Th - #r Ala Gly Asp Thr His     #   220     - Leu Gly Gly Glu Asp Phe Asp Asn Arg Leu Va - #l Asn His Phe Ile Gln     225                 2 - #30                 2 - #35                 2 -     #40     - Glu Phe Lys Arg Lys Asn Lys Lys Asp Leu Se - #r Thr Asn Gln Arg Ala     #               255     - Leu Arg Arg Leu Arg Thr Ala Cys Glu Ser Gl - #n Glu Asn Phe Val Ser     #           270     - Ser Ala Gln Thr Ser Val Glu Ile Asp Ser Ly - #s Asn Glu Gly Ile Asp     #       285     - Phe Tyr Thr Ser Ile Thr Arg Ala Arg Phe Gl - #u Glu Leu Cys Ala Asp     #   300     - Leu Phe Arg Ser Thr Leu Asp Pro Val Glu Ly - #s Val Leu Arg Asp Ala     305                 3 - #10                 3 - #15                 3 -     #20     - Lys Leu Asp Lys Ser Gln Val Asp Glu Ile Va - #l Leu Val Gly Gly Ser     #               335     - Thr Arg Ile     - (2) INFORMATION FOR SEQ ID NO:53:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 560 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: None     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:     - Lys Thr Leu Pro Phe Ser Glu Asn Phe Ile Me - #t Ala Asp Ser Glu Glu     #                15     - Tyr Lys Thr Val Ile Gly Ile Ser Phe Gly As - #n Gln Asn Ser Ser Ile     #            30     - Ala Phe Asn Arg Asp Gly Lys Thr Asp Val Le - #u Ala Asn Glu Glu Gly     #        45     - Asn Arg Gln Ile Pro Ser Ile Leu Pro Tyr Hi - #s Gly Asp Gln Glu Tyr     #    60     - His Gly Val Gln Ala Arg Gly Gln Leu Val Ar - #g Asn Ala Asp Asn Ser     #80     - Val Thr Asn Phe Arg Asp Leu Leu Gly Lys Se - #r His Asp Glu Leu Thr     #                95     - His His His Cys His Tyr Ser Ser Asn Pro Va - #l Asn Val Glu Gly Gln     #           110     - Ile Gly Phe Lys Ile Thr Val Gln Glu Gly Gl - #u Glu Ser Asp Pro Lys     #       125     - Glu Lys Ile Leu Thr Ala His Glu Ala Ser Va - #l Arg His Leu Arg Arg     #   140     - Leu Thr Glu Ser Ala Glu Asp Phe Leu Gly Th - #r Lys Val Asn Gly Cys     145                 1 - #50                 1 - #55                 1 -     #60     - Val Met Ser Val Pro Val Tyr Phe Thr Asp Al - #a Gln Arg Lys Ala Leu     #               175     - Glu Ser Ala Ala Asn Glu Ala Gly Leu Pro Va - #l Leu Gln Leu Ile His     #           190     - Asp Pro Ala Ala Val Ile Leu Ala Leu Met Ty - #r Ser Glu Glu Val Leu     #       205     - Ile Asp Lys Thr Val Val Val Ala Asn Phe Gl - #y Ala Thr Arg Ser Glu     #   220     - Val Ser Val Val Ser Val Lys Gly Gly Leu Me - #t Thr Ile Leu Ala Ser     225                 2 - #30                 2 - #35                 2 -     #40     - Val His Asp Glu Asn Leu Gly Gly Glu Gln Le - #u Thr Asp Val Leu Val     #               255     - Asn Phe Phe Ala Lys Glu Phe Glu Lys Lys As - #n Gly Ile Asp Pro Arg     #           270     - Lys Asn Ala Arg Ser Leu Val Lys Leu Arg Al - #a Gln Cys Glu Ile Thr     #       285     - Lys Arg Val Leu Ser Asn Gly Thr Thr Ala Se - #r Ala Ala Val Asp Ser     #   300     - Leu Ala Asp Gly Ile Asp Phe His Ser Ser Il - #e Asn Arg Leu Arg Tyr     305                 3 - #10                 3 - #15                 3 -     #20     - Asp Leu Ala Ala Ser Ala Thr Leu Asn Arg Me - #t Ala Asp Leu Val Thr     #               335     - Glu Ala Val Glu Lys Ala Asn Met Glu Pro Ph - #e Asp Ile Ser Glu Val     #           350     - Ile Leu Ala Gly Gly Ala Ser Asn Thr Pro Ly - #s Leu Thr Ser Leu Met     #       365     - Glu Ser Ile Phe Pro Glu Gln Thr Ile Ile Ar - #g Ser Ser Ser Ser Val     #   380     - Thr Pro Leu Gln Leu Asp Pro Ser Glu Leu Th - #r Ala Ile Gly Ser Gly     385                 3 - #90                 3 - #95                 4 -     #00     - Val Gln Ala Ser Leu Ile Gly His Phe Asp Al - #a Ala Asp Ile Ala Ala     #               415     - Ser Thr Asp Ala Gln Val Val Asp Val Pro Hi - #s Leu Thr Ala Pro Ile     #           430     - Gly Ile Asn Glu Gly Glu Asn Phe Val Thr Il - #e Phe Asp Ile Glu Thr     #       445     - Ala Leu Pro Ala Arg Lys Thr Val Glu Val Il - #e Ala Pro Lys Glu Gly     #   460     - Ala Ala Phe Ile Pro Ile Tyr Glu Ala Glu Ar - #g Ser Val Lys Val Thr     465                 4 - #70                 4 - #75                 4 -     #80     - Lys Val Glu Pro Glu Pro Ile Asp Glu Glu Gl - #u Ala Phe Ser Asp Asp     #               495     - Glu Glu Glu Glu Pro Glu Glu Ile Lys Glu Ar - #g Ile Ala Ile Pro Lys     #           510     - Thr Leu Ile Ala Thr Ile Thr Leu Pro Asp Va - #l Ser Pro Asn Ala Lys     #       525     - Ile Glu Leu Val Leu Gln Ile Asp Ala Glu Gl - #y Lys Leu Thr Ala Ser     #   540     - Ala Arg Pro Lys Asp Gly Lys Gly Thr Asn Va - #l Arg Gly Ser Thr Ala     545                 5 - #50                 5 - #55                 5 -     #60     - (2) INFORMATION FOR SEQ ID NO:54:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:     #  18              GC     - (2) INFORMATION FOR SEQ ID NO:55:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:     #  18              GC     - (2) INFORMATION FOR SEQ ID NO:56:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 501 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:     - ATTTCCCAGC GTCGTCGTTG GTTGAACGGT TCTTTTGCGG CCGGTCTCTA TT - #CGCTCATG       60     - CATTTCGGTC GGATGTACAA GAGTGGACAT AACATCATCC GTATGTTCTT CT - #TGCACATT      120     - CAGATGTTGT ACAACGTTTT CAACACTATC CTTACATGGT TCTCCCTGGC AT - #CTTACTGG      180     - TTGACCACCA CCGTCATCAT GGACTTGGTC GGAACGCCCA GTGAGAGCAA CG - #GTAACAAA      240     - GGATTCCCCT TCGGTAAATC GGCGACCCCT ATTATCAACA CAATTGTGAA GT - #ATGTCTAC      300     - CTCGGATTGT TGCTCTTGCA GTTCATTCTC GCTCTCGGTA ACCGCCCCAA GG - #GATCCCGC      360     - TTCTCGTACC TGACATCTTT CGTCGTATTC GGTATCATTC AAATCTACGT TG - #TCGTCGAC      420     - GCTCTGTACT TGGTGGTTCG TGCATTCAAG GTGTTGGCGA ATTCCTCAAG TC - #GTTCTTCT      480     #                 501CC A     - (2) INFORMATION FOR SEQ ID NO:57:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 178 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: None     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:     - Ile Ser Gln Arg Arg Arg Trp Leu Asn Gly Se - #r Phe Ala Ala Gly Leu     #                15     - Tyr Ser Leu Met His Phe Gly Arg Met Tyr Ly - #s Ser Gly His Asn Ile     #            30     - Ile Arg Met Phe Phe Leu His Ile Gln Met Le - #u Tyr Asn Val Phe Asn     #        45     - Thr Ile Leu Thr Trp Phe Ser Leu Ala Ser Ty - #r Trp Leu Thr Thr Thr     #    60     - Val Ile Met Asp Leu Val Gly Thr Pro Ser Gl - #u Ser Asn Gly Asn Lys     #80     - Gly Phe Pro Phe Gly Lys Ser Ala Thr Pro Il - #e Ile Asn Thr Ile Val     #                95     - Lys Tyr Val Tyr Leu Gly Leu Leu Leu Leu Gl - #n Phe Ile Leu Ala Leu     #           110     - Gly Asn Arg Pro Lys Gly Ser Arg Phe Ser Ty - #r Leu Thr Ser Phe Val     #       125     - Val Phe Gly Ile Ile Gln Ile Tyr Val Val Va - #l Asp Ala Leu Tyr Leu     #   140     - Val Val Arg Ala Phe Thr Asn Ser Asp Ala Il - #e Asp Phe Val Thr Asp     145                 1 - #50                 1 - #55                 1 -     #60     - Gln Gly Val Gly Glu Phe Leu Lys Ser Phe Ph - #e Ser Ser Ser Gly Ala     #               175     - Ser Ala     - (2) INFORMATION FOR SEQ ID NO:58:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 916 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: None     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:     - Met Ala Tyr His Gly Ser Gly Pro Gln Ser Pr - #o Gly Glu His Thr Tyr     #                15     - Asp Asp Gly His Gln Leu Arg Asp Leu Ser Hi - #s Ser Asn Thr Ser Tyr     #            30     - Glu Glu Glu Ala Ser His Gly Leu Leu Ser Se - #r Gln Gln Ser Pro Phe     #        45     - Ala Gly Pro Phe Asp Asp Pro His Gln Gln Ar - #g Gly Leu Thr Ala Ser     #    60     - Pro Val Gln Arg Pro Thr Ser Gly Tyr Ser Le - #u Thr Glu Ser Tyr Ala     #80     - Pro Asp Ala Ala Tyr His Asp Pro Tyr Ser Al - #a Asn Gln Ser Val Tyr     #                95     - Ser Gly His Ser Glu Asn Pro Ala Ala Ala Ph - #e Gly Val Pro Gly Arg     #           110     - Val Ala Ser Pro Tyr Ala Arg Ser Glu Thr Se - #r Ser Thr Glu Ala Trp     #       125     - Arg Gln Arg Gln Ala Gly Ala Arg Arg Gly Gl - #y Asn Gly Leu Arg Arg     #   140     - Tyr Ala Thr Arg Lys Val Lys Leu Val Gln Gl - #y Ser Val Leu Ser Val     145                 1 - #50                 1 - #55                 1 -     #60     - Asp Tyr Pro Val Pro Ser Ala Ile Gln Asn Al - #a Ile Gln Ala Lys Tyr     #               175     - Arg Asn Asp Leu Glu Gly Gly Ser Glu Glu Ph - #e Thr His Met Arg Tyr     #           190     - Thr Ala Ala Thr Cys Asp Pro Asn Glu Phe Th - #r Leu His Asn Gly Tyr     #       205     - Asn Leu Arg Pro Ala Met Tyr Asn Arg His Th - #r Glu Leu Leu Ile Ala     #   220     - Ile Thr Tyr Tyr Asn Glu Asp Lys Thr Leu Th - #r Ala Arg Thr Leu His     225                 2 - #30                 2 - #35                 2 -     #40     - Gly Val Met Gln Asn Ile Arg Asp Ile Val As - #n Leu Lys Lys Ser Glu     #               255     - Phe Trp Asn Lys Gly Gly Pro Ala Trp Gln Ly - #s Ile Val Val Cys Leu     #           270     - Val Phe Asp Gly Ile Asp Pro Cys Asp Lys As - #p Thr Leu Asp Val Leu     #       285     - Ala Thr Val Gly Ile Tyr Gln Asp Gly Val Me - #t Lys Arg Asp Val Asp     #   300     - Gly Lys Glu Thr Val Ala His Ile Phe Glu Ty - #r Thr Thr Gln Leu Ser     305                 3 - #10                 3 - #15                 3 -     #20     - Val Thr Pro Asn Gln Gln Leu Ile Arg Pro Th - #r Asp Asp Gly Pro Ser     #               335     - Thr Leu Pro Pro Val Gln Met Met Phe Cys Le - #u Lys Gln Lys Asn Ser     #           350     - Lys Lys Ile Asn Ser His Arg Trp Leu Phe As - #n Ala Phe Gly Arg Ile     #       365     - Leu Asn Pro Glu Val Cys Ile Leu Leu Asp Al - #a Gly Thr Lys Pro Gly     #   380     - Pro Lys Ser Leu Leu Tyr Leu Trp Glu Ala Ph - #e Tyr Asn Asp Lys Asp     385                 3 - #90                 3 - #95                 4 -     #00     - Leu Gly Gly Ala Cys Gly Glu Ile His Ala Me - #t Leu Gly Lys Gly Trp     #               415     - Lys Lys Leu Leu Asn Pro Leu Val Ala Ala Gl - #n Asn Phe Glu Tyr Lys     #           430     - Ile Ser Asn Ile Leu Asp Lys Pro Leu Glu Se - #r Ser Phe Gly Tyr Val     #       445     - Ser Val Leu Pro Gly Ala Phe Ser Ala Tyr Ar - #g Phe Arg Ala Ile Met     #   460     - Gly Arg Pro Leu Glu Gln Tyr Phe His Gly As - #p His Thr Leu Ser Lys     465                 4 - #70                 4 - #75                 4 -     #80     - Gln Leu Gly Lys Lys Gly Ile Glu Gly Met As - #n Ile Phe Lys Lys Asn     #               495     - Met Phe Leu Ala Glu Asp Arg Ile Leu Cys Ph - #e Glu Leu Val Ala Lys     #           510     - Ala Gly Ser Lys Trp His Leu Ser Tyr Val Ly - #s Ala Ser Lys Gly Glu     #       525     - Thr Asp Val Pro Glu Gly Ala Pro Glu Phe Il - #e Ser Gln Arg Arg Arg     #   540     - Trp Leu Asn Gly Ser Phe Ala Ala Gly Ile Ty - #r Ser Leu Met His Phe     545                 5 - #50                 5 - #55                 5 -     #60     - Gly Arg Met Tyr Lys Ser Gly His Asn Ile Va - #l Arg Met Phe Phe Leu     #               575     - His Leu Gln Met Leu Tyr Asn Trp Phe Ser Th - #r Phe Leu Thr Trp Phe     #           590     - Ser Leu Ala Ser Tyr Trp Leu Thr Thr Ser Va - #l Ile Met Asp Leu Val     #       605     - Gly Thr Pro Ser Ser Ser Asn Gly Tyr Thr Al - #a Phe Pro Phe Gly Lys     #   620     - Thr Ala Thr Pro Ile Ile Asn Thr Leu Val Ly - #s Tyr Ile Tyr Leu Ala     625                 6 - #30                 6 - #35                 6 -     #40     - Phe Leu Leu Leu Gln Phe Ile Leu Ala Leu Gl - #y Asn Arg Pro Lys Gly     #               655     - Ser Lys Leu Ser Tyr Leu Ala Ser Phe Val Al - #a Phe Gly Ile Ile Gln     #           670     - Leu Tyr Val Val Val Asp Ala Leu Tyr Leu Va - #l Val Arg Ala Phe Thr     #       685     - Gly Gly Ala Pro Met Asp Phe Asn Thr Asp As - #p Gly Ile Gly Ala Phe     #   700     - Leu Ser Ser Phe Phe Gly Ser Ser Gly Ala Gl - #y Ile Ile Ile Ile Ala     705                 7 - #10                 7 - #15                 7 -     #20     - Leu Ala Ala Thr Phe Gly Leu Tyr Phe Val Al - #a Ser Phe Met Tyr Leu     #               735     - Asp Pro Trp His Met Phe Thr Ser Phe Pro Al - #a Tyr Met Ala Val Gln     #           750     - Ser Ser Tyr Ile Asn Ile Leu Asn Val Tyr Al - #a Phe Ser Asn Trp His     #       765     - Asp Val Ser Trp Gly Thr Lys Gly Ser Asp Ly - #s Ala Asp Ala Leu Pro     #   780     - Ser Ala Lys Thr Thr Gly Gly Lys Gly Glu Gl - #u Ala Val Ile Glu Glu     785                 7 - #90                 7 - #95                 8 -     #00     - Ile Asp Lys Pro Gln Ala Asp Ile Asp Ser Gl - #n Phe Glu Ala Thr Val     #               815     - Lys Arg Ala Leu Thr Pro Tyr Val Pro Pro Gl - #u Glu Lys Glu Glu Lys     #           830     - Ser Leu Asp Asp Ser Tyr Lys Ser Phe Arg Th - #r Arg Leu Val Thr Leu     #       845     - Trp Leu Phe Ser Asn Gly Leu Leu Ala Val Cy - #s Ile Thr Ser Glu Gly     #   860     - Leu Asp Lys Phe Gly Phe Thr Asn Thr Ser Th - #r Glu Arg Thr Ser Arg     865                 8 - #70                 8 - #75                 8 -     #80     - Phe Phe Gln Ala Leu Leu Trp Ser Asn Ala Va - #l Val Ala Leu Ile Arg     #               895     - Phe Ile Gly Ala Thr Trp Phe Leu Gly Lys Th - #r Gly Leu Leu Cys Cys     #           910     - Phe Ala Arg Arg             915     - (2) INFORMATION FOR SEQ ID NO:59:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 911 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: None     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:     - Met Ala Tyr Gln Gly Ser Gly Ser His Ser Pr - #o Pro His Tyr Asp Asp     #                15     - Asn Gly His Arg Leu Gln Asp Leu Pro His Gl - #y Ser Tyr Glu Glu Glu     #            30     - Ala Ser Arg Gly Leu Leu Ser His Gln Gln Gl - #y Pro Phe Thr Gly Pro     #        45     - Phe Asp Asp Pro Gln Gln His Gly Ser Ser Th - #r Thr Arg Pro Val Ser     #    60     - Gly Tyr Ser Leu Ser Glu Thr Tyr Ala Pro Gl - #u Ala Ala Tyr His Asp     #80     - Pro Tyr Thr Gln Pro Ser Pro Gly Ser Val Ty - #r Ser Ala Gln Ser Ala     #                95     - Glu Asn Pro Ala Ala Ala Phe Gly Val Pro Gl - #y Arg Val Ala Ser Pro     #           110     - Tyr Ala Arg Ser Asp Thr Ser Ser Thr Glu Al - #a Trp Arg Gln Arg Gln     #       125     - Ala Pro Gly Gly Gly Pro Gly Gly Leu Arg Ar - #g Tyr Ala Thr Arg Lys     #   140     - Val Lys Leu Val Gln Gly Ser Val Leu Ser Va - #l Asp Tyr Pro Val Pro     145                 1 - #50                 1 - #55                 1 -     #60     - Ser Ala Ile Gln Asn Ala Ile Gln Ala Lys Ty - #r Arg Asn Asp Leu Glu     #               175     - Gly Gly Ser Glu Glu Phe Thr His Met Arg Ty - #r Thr Ala Ala Thr Cys     #           190     - Asp Pro Asn Glu Phe Thr Leu His Asn Gly Ty - #r Asn Leu Arg Pro Ala     #       205     - Met Tyr Asn Arg His Thr Glu Leu Leu Ile Al - #a Ile Thr Tyr Tyr Asn     #   220     - Glu Asp Lys Thr Leu Thr Ser Arg Thr Leu Hi - #s Gly Val Met Gln Asn     225                 2 - #30                 2 - #35                 2 -     #40     - Ile Arg Asp Ile Val Asn Leu Lys Lys Ser Gl - #u Phe Trp Asn Lys Gly     #               255     - Gly Pro Ala Trp Gln Lys Ile Val Val Cys Le - #u Val Phe Asp Gly Ile     #           270     - Asp Pro Cys Asp Lys Asp Thr Leu Asp Val Le - #u Ala Thr Ile Gly Val     #       285     - Tyr Gln Asp Gly Val Met Lys Arg Asp Val As - #p Gly Lys Glu Thr Val     #   300     - Ala His Ile Phe Glu Tyr Thr Thr Gln Leu Se - #r Val Thr Pro Asn Gln     305                 3 - #10                 3 - #15                 3 -     #20     - Gln Leu Ile Arg Pro Thr Asp Asp Gly Pro Se - #r Thr Leu Leu Pro Ser     #               335     - Lys Met Met Phe Cys Leu Lys Gln Lys Asn Se - #r Lys Lys Ile Asn Ser     #           350     - His Arg Trp Leu Phe Asn Ala Phe Gly Arg Il - #e Leu Asn Pro Glu Val     #       365     - Cys Ile Leu Leu Asp Ala Gly Thr Lys Pro Gl - #y Pro Lys Ser Leu Leu     #   380     - Ser Leu Trp Glu Ala Phe Tyr Asn Asp Lys As - #p Leu Gly Gly Ala Cys     385                 3 - #90                 3 - #95                 4 -     #00     - Gly Glu Ile His Ala Met Leu Gly Lys Gly Tr - #p Lys Asn Leu Ile Asn     #               415     - Pro Leu Val Ala Ala Gln Asn Phe Glu Tyr Ly - #s Ile Ser Asn Ile Leu     #           430     - Asp Lys Pro Leu Glu Ser Ser Phe Gly Tyr Va - #l Ser Val Leu Pro Gly     #       445     - Ala Phe Ser Ala Tyr Arg Phe Arg Ala Ile Me - #t Gly Arg Pro Leu Glu     #   460     - Gln Tyr Phe His Gly Asp His Thr Leu Ser Ly - #s Gln Leu Gly Lys Lys     465                 4 - #70                 4 - #75                 4 -     #80     - Gly Ile Glu Gly Met Asn Ile Phe Lys Lys As - #n Met Phe Leu Ala Glu     #               495     - Asp Arg Ile Leu Cys Phe Glu Leu Val Ala Ly - #s Ala Gly Ser Lys Trp     #           510     - His Leu Thr Tyr Val Lys Ala Ser Lys Ala Gl - #u Thr Asp Val Pro Glu     #       525     - Gly Ala Pro Glu Phe Ile Ser Gln Arg Arg Ar - #g Trp Leu Asn Gly Ser     #   540     - Phe Ala Ala Gly Ile Tyr Ser Leu Met His Ph - #e Gly Arg Met Tyr Lys     545                 5 - #50                 5 - #55                 5 -     #60     - Ser Gly His Asn Ile Val Arg Met Phe Phe Le - #u His Ile Gln Met Leu     #               575     - Tyr Asn Ile Phe Ser Thr Val Leu Thr Trp Ph - #e Ser Leu Ala Ser Tyr     #           590     - Trp Leu Thr Thr Thr Val Ile Met Asp Leu Va - #l Gly Thr Pro Ser Asp     #       605     - Asn Asn Gly Asn Lys Ala Phe Pro Phe Gly Ly - #s Thr Ala Thr Pro Ile     #   620     - Ile Asn Thr Ile Val Lys Tyr Val Tyr Leu Gl - #y Phe Leu Leu Leu Gln     625                 6 - #30                 6 - #35                 6 -     #40     - Phe Ile Leu Ala Leu Gly Asn Arg Pro Lys Gl - #y Ser Lys Phe Ser Tyr     #               655     - Leu Ala Ser Phe Val Val Phe Gly Ile Ile Gl - #n Val Tyr Val Val Ile     #           670     - Asp Ala Leu Tyr Leu Val Val Arg Ala Phe Se - #r Gly Ser Ala Pro Met     #       685     - Asp Phe Thr Thr Asp Gln Gly Val Gly Glu Ph - #e Leu Lys Ser Phe Phe     #   700     - Ser Ser Ser Gly Ala Gly Ile Ile Ile Ile Al - #a Leu Ala Ala Thr Phe     705                 7 - #10                 7 - #15                 7 -     #20     - Gly Leu Tyr Phe Val Ala Ser Phe Met Tyr Le - #u Asp Pro Trp His Met     #               735     - Phe Thr Ser Phe Pro Ala Tyr Met Cys Val Gl - #n Ser Ser Tyr Ile Asn     #           750     - Ile Leu Asn Val Tyr Ala Phe Ser Asn Trp Hi - #s Asp Val Ser Trp Gly     #       765     - Thr Lys Gly Ser Asp Lys Ala Asp Ala Leu Pr - #o Ser Ala Lys Thr Thr     #   780     - Lys Asp Glu Gly Lys Glu Val Val Ile Glu Gl - #u Ile Asp Lys Pro Gln     785                 7 - #90                 7 - #95                 8 -     #00     - Ala Asp Ile Asp Ser Gln Phe Glu Ala Thr Va - #l Lys Arg Ala Leu Thr     #               815     - Pro Tyr Val Pro Pro Val Glu Lys Glu Glu Ly - #s Thr Leu Glu Asp Ser     #           830     - Tyr Lys Ser Phe Arg Thr Arg Leu Val Thr Ph - #e Trp Ile Phe Ser Asn     #       845     - Ala Phe Leu Ala Val Cys Ile Thr Ser Asp Gl - #y Val Asp Lys Phe Gly     #   860     - Phe Thr Asn Ser Ala Thr Asp Arg Thr Gln Ar - #g Phe Phe Gln Ala Leu     865                 8 - #70                 8 - #75                 8 -     #80     - Leu Trp Ser Asn Ala Val Val Ala Leu Phe Ar - #g Phe Ile Gly Ala Cys     #               895     - Trp Phe Leu Gly Lys Thr Gly Leu Met Cys Cy - #s Phe Ala Arg Arg     #           910     - (2) INFORMATION FOR SEQ ID NO:60:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 17 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:     #   17             C     - (2) INFORMATION FOR SEQ ID NO:61:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 17 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:     #   17             A     - (2) INFORMATION FOR SEQ ID NO:62:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 21 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:     #21                CGCA T     - (2) INFORMATION FOR SEQ ID NO:63:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 251 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:     - AGGGCCACAG CNTGTGCTAA GCGCCTTGAC GGGGACCCTG GGACTTTCAA GT - #CTCCTTGG       60     - ACCCGGAATT GAATCCTCAC AGAACAGCTT TCAACACTGC TCTAAGGCTG AA - #CTGAGCTG      120     - CGCGACTCCG TATCATGGCC AAGACAAATG CTGCTTCAAC TATCCCGGGG GG - #CAGTTCCT      180     - TCAATCGCTG TTTTGGGACG CCGACCCGGC CATTGGACCG GAAGATTCCT GG - #ACTATCCA      240     #      251     - (2) INFORMATION FOR SEQ ID NO:64:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 83 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: None     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:     - Gly Pro Gln Xaa Val Leu Ser Ala Leu Thr Gl - #y Thr Leu Gly Leu Ser     #                15     - Ser Leu Leu Gly Pro Gly Ile Glu Ser Ser Gl - #n Asn Ser Phe Gln His     #            30     - Cys Ser Lys Ala Glu Leu Ser Cys Ala Thr Pr - #o Tyr His Gly Gln Asp     #        45     - Lys Cys Cys Phe Asn Tyr Pro Gly Gly Gln Ph - #e Leu Gln Ser Leu Phe     #    60     - Trp Asp Ala Asp Pro Ala Ile Gly Pro Glu As - #p Ser Trp Thr Ile His     #80     - Gly Leu Trp     - (2) INFORMATION FOR SEQ ID NO:65:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 83 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: None     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:     - Gly Pro Gln Xaa Val Leu Ser Ala Leu Thr Gl - #y Thr Leu Gly Leu Ser     #                15     - Ser Leu Leu Gly Pro Gly Ile Glu Ser Ser Gl - #n Asn Ser Phe Gln His     #            30     - Cys Ser Lys Ala Glu Leu Ser Cys Ala Thr Pr - #o Tyr His Gly Gln Asp     #        45     - Lys Cys Cys Phe Asn Tyr Pro Gly Gly Gln Ph - #e Leu Gln Ser Leu Phe     #    60     - Trp Asp Ala Asp Pro Ala Ile Gly Pro Glu As - #p Ser Trp Thr Ile His     #80     - Gly Leu Trp     - (2) INFORMATION FOR SEQ ID NO:66:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 214 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:     - CTACTGAACG CTTAAAGGTG CTTAAGGAGC AACTTCATAT TATGCGCGAC CA - #ACGGATCC       60     - AGGAAGTCTT GAGCAATAAG AAGGGTCGAA CGCAGCACGG ACACTCGCAC AA - #GCCGACCG      120     - GTTTTGGGGG ACTCAACGGT TCTCGGCTAA AGGAGGCCTT TGTGGGACGT CG - #AATCGGGA      180     #       214        GGCC GAATTGGCCA CCCC     - (2) INFORMATION FOR SEQ ID NO:67:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:     #  18              CG     - (2) INFORMATION FOR SEQ ID NO:68:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:     #  18              TG     - (2) INFORMATION FOR SEQ ID NO:69:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:     #  18              AG     - (2) INFORMATION FOR SEQ ID NO:70:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:     #  18              AG     - (2) INFORMATION FOR SEQ ID NO:71:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 336 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:     - AGCACCTATA ATCTATGCTG TCCCACTATC ACACATCTAT ATGTTGTACA AG - #CCTGATAC       60     - AATCAATAAT GATGTAATAA TTGACTCTGG AAAGTTGGCT ATAAAACTCA CC - #ATACAAGT      120     - CCAGATAACC CTGCCAAACT CCACTCCCAG GGCATTAATC TTCATTTATA TC - #GACCAGCC      180     - ATACCTATGG TCAAATCACA CGCAACGCCA CAGATATATA TTTGAATCAA AT - #TTCTCTTT      240     - TGAAGAAGAA AGGGTGGTTT ATGAGGAAGA ATATCCCAAT ATGCCAATCT GA - #CTGTTCCG      300     #      336         AAGC TTCGAATATA AATATA     - (2) INFORMATION FOR SEQ ID NO:72:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:     #  18              CC     - (2) INFORMATION FOR SEQ ID NO:73:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:     #  18              CC     - (2) INFORMATION FOR SEQ ID NO:74:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:     #  18              TC     - (2) INFORMATION FOR SEQ ID NO:75:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:     #  18              TG     - (2) INFORMATION FOR SEQ ID NO:76:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 281 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:     - CAAGGGAACG GGAATAAAAT ACACATAACA AAGGATTCGA AGAAAGAAAA AA - #AAAGGGGG       60     - GGAGGTGTGT CCAAGAGGAA AGAAGAAAAA AAATTTAATT TCGCCACCCT AT - #CGCGGAGT      120     - GTTCCGCCTT CAGGAGAGAT AGAAAAGAGG AGGGAGAAGG GAGAAGGAAA AA - #AAAAAACA      180     - GAATTCCCAC AGACAAGGAA AGCTTAACCG GGTCACGAAA AAGCACAATA CA - #GGTGAACA      240     #  281             CCAA AAAGAAAAAA ATAATTCCTA A     - (2) INFORMATION FOR SEQ ID NO:77:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:     #  18              TG     - (2) INFORMATION FOR SEQ ID NO:78:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:     #  18              GG     - (2) INFORMATION FOR SEQ ID NO:79:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:     #  18              GC     - (2) INFORMATION FOR SEQ ID NO:80:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 18 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:     #  18              AC     __________________________________________________________________________ 

What is claimed is:
 1. A method of producing a polypeptide, comprising:(a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein(i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, not within a second DNA sequence encoding a protein that negatively regulates transcription, translation or secretion of the polypeptide, and not within a third DNA sequence encoding a protease which hydrolyzes the polypeptide under the conditions; and (ii) the mutant cell produces more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and (b) recovering the polypeptide.
 2. The method of claim 1, wherein the nucleic acid construct cannot homologous recombine with the first DNA sequence.
 3. The method of claim 1, wherein the nucleic acid construct cannot homologous recombine with the locus.
 4. The method of claim 1, wherein the locus is on a different chromosome than the first DNA sequence or on the same chromosome but at least 3,000 bps from the 5' or 3' terminus of the first DNA sequence.
 5. The method of claim 1, wherein the nucleic acid construct is introduced by restriction enzyme-mediated integration.
 6. The method of claim 1, wherein the nucleic acid construct comprises a selectable marker.
 7. The method of claim 6, wherein the selectable marker is amdS, argB, bar, hygB, niaD, pyrG, sC, or trpC.
 8. The method of claim 1, wherein the parent cell is a mammalian cell.
 9. The method of claim 1, wherein the parent cell is a bacterial cell.
 10. The method of claim 1, wherein the parent cell is a fungal cell.
 11. The method of claim 10, wherein the fungal cell is a filamentous fungal cell.
 12. The method of claim 11, wherein the filamentous fungal cell is selected from the group consisting of Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia, Tolypocladium, and Trichoderma.
 13. The method of claim 10, wherein the fungal cell is a yeast cell.
 14. The method of claim 1, wherein the polypeptide is a recombinant polypeptide.
 15. The method of claim 1, wherein the polypeptide is a heterologous polypeptide.
 16. The method of claim 1, wherein the polypeptide is a hormone, enzyme, receptor or portions thereof, antibody or portions thereof, or reporter.
 17. The method of claim 16, wherein the enzyme is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase.
 18. The method of claim 17, wherein the polypeptide is an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, or xylanase.
 19. The method of claim 1, wherein the mutant cell has an increased uptake of an inorganic cofactor compared to the parent cell.
 20. The method of claim 1, wherein the mutant cell has a more desirable morphology than the parent cell.
 21. The method of claim 1, wherein the mutant cell produces higher yields of one or more secreted proteins than the parent cell.
 22. The method of claim 1, wherein the mutant cell which has lost its ability to synthesize one or more essential metabolites.
 23. The method of claim 1, wherein a phenotype of the mutant cell is observed only under certain conditions.
 24. The method of claim 1, wherein the mutant cell exhibits an altered growth rate relative to the parent cell.
 25. The method of claim 1, wherein the growth of the mutant cell is not inhibited by the overproduction of a desired polypeptide or metabolite when grown under conditions that induce high level production of the polypeptide or metabolite.
 26. The method of claim 1, wherein the mutant cell is able to tolerate lower oxygen conditions than the parent cell.
 27. The method of claim 1, wherein the mutant cell exhibits altered production of a transcriptional activator of a promoter than the parent cell.
 28. The method of claim 1, wherein the mutant cell has a mutation in one or more of the genes of a signal transduction pathway of the parent cell.
 29. The method of claim 1, wherein the mutant cell does not erroneously splice a cryptic intron.
 30. The method of claim 1, wherein the nucleic acid construct is pDSY109, pDSY112, pMT1936, pDSY138, pDSY162, pDSY163, pDSY141, pSMO1204, pSMOH603, p4-8.1, p7-14.1, pHB220, pSMO717, pSMO321, pHowB571 or pSMO810.
 31. The method of claim 1, wherein the locus is SEQ ID NO:9, SEQ ID NO:16, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:50, SEQ ID NO:56, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:71, SEQ ID NO:76, or a fragment thereof.
 32. The method of claim 1, wherein the locus encodes a glucose transporter, mannitol-1-phosphate dehydrogenase, chitin synthase, heat shock protein, manganese superoxide dismutase, or a gene required for activation of pacC.
 33. The method of claim 32, where the gene required for activation of pacC is a palB gene.
 34. A method of producing a polypeptide, comprising(a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein(i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, wherein the introduction of the nucleic acid construct disrupts a gene encoding an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, or regulatory or control sequences thereof, other than a gene encoding a protease which hydrolyzes the polypeptide under the conditions; and (ii) the mutant cell produces more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and (b) recovering the polypeptide.
 35. A method of producing a polypeptide, comprising(a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein(i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, not within a second DNA sequence encoding a protein that negatively regulates transcription of the polypeptide, and not within a third DNA sequence encoding a protease which hydrolyzes the polypeptide under the conditions; and (ii) the mutant cell expresses more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and (b) recovering the polypeptide.
 36. The method of claim 35 wherein the nucleic acid construct cannot homologous recombine with the first DNA sequence.
 37. The method of claim 35, wherein the nucleic acid construct cannot homologous recombine with the locus.
 38. The method of claim 35, wherein the locus is on a different chromosome than the first DNA sequence or on the same chromosome but at least 3,000 bps from the 5' or 3' terminus of the first DNA sequence.
 39. A method of producing a polypeptide, comprising(a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein(i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, wherein the introduction of the nucleic acid construct disrupts a gene encoding an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, or regulatory or control sequences thereof, other than a gene encoding a protease which hydrolyzes the polypeptide under the conditions; and (ii) the mutant cell expresses more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and (b) recovering the polypeptide.
 40. A method of producing a polypeptide, comprising(a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein(i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, not within a second DNA sequence encoding a protein that negatively regulates translation of the polypeptide, and not within a third DNA sequence encoding a protease which hydrolyzes the polypeptide under the conditions; and (ii) the mutant cell synthesizes more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and (b) recovering the polypeptide.
 41. The method of claim 40, wherein the nucleic acid construct cannot homologous recombine with the first DNA sequence.
 42. The method of claim 40, wherein the nucleic acid construct cannot homologous recombine with the locus.
 43. The method of claim 40, wherein the locus is on a different chromosome than the first DNA sequence or on the same chromosome but at least 3,000 bps from the 5' or 3' terminus of the first DNA sequence.
 44. A method of producing a polypeptide, comprising(a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein(i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, wherein the introduction of the nucleic acid construct disrupts a gene encoding an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, or regulatory or control sequences thereof, other than a gene encoding a protease which hydrolyzes the polypeptide under the conditions; and (ii) the mutant cell synthesizes more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and (b) recovering the polypeptide.
 45. A method of producing a polypeptide, comprising(a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein(i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, not within a second DNA sequence encoding a protein that negatively regulates secretion of the polypeptide, and not within a third DNA sequence encoding a protease which hydrolyzes the polypeptide under the conditions; and (ii) the mutant cell secretes more of the polypeptide than the parent cell when both cells are cultivated under the conditions; (b) recovering the polypeptide.
 46. The method of claim 45, wherein the nucleic acid construct cannot homologous recombine with the first DNA sequence.
 47. The method of claim 45, wherein the nucleic acid construct cannot homologous recombine with the locus.
 48. The method of claim 45, wherein the locus is on a different chromosome than the first DNA sequence or on the same chromosome but at least 3,000 bps from the 5' or 3' terminus of the first DNA sequence.
 49. A method of producing a polypeptide, comprising(a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein(i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence, wherein the introduction of the nucleic acid construct disrupts a gene encoding an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, or regulatory or control sequences thereof, other than a gene encoding a protease which hydrolyzes the polypeptide under the conditions; and (ii) the mutant cell secretes more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and (b) recovering the polypeptide.
 50. A method of producing a polypeptide, comprising(a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein(i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the random integration of a nucleic acid construct into the genome of the parent cell at a locus wherein the nucleic acid construct is not homologous with the locus and wherein the locus is not within the first DNA sequence nor within a second DNA sequence encoding a protease which hydrolyzes the polypeptide under the conditions; and (ii) the mutant cell produces more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and (b) recovering the polypeptide.
 51. A method of producing a metabolite, comprising(A) cultivating a mutant cell under conditions conducive for production of the metabolite, wherein(i) the mutant cell is related to a parent cell, which comprises one or more first DNA sequences encoding first polypeptides in the biosynthetic pathway of the metabolite, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within (a) the first DNA sequences, (b) a second DNA sequence encoding a protein that negatively regulates transcription, translation or secretion of the first polypeptides, (c) a third DNA sequence encoding a protease which hydrolyzes any of the first polypeptides under the conditions, and (d) one or more fourth DNA sequences encoding a second polypeptide in the second biosynthetic pathway of a second metabolite wherein the biosynthetic pathway and the second biosynthetic pathway involve the production of the same intermediate and the second polypeptide catalyzes a step after the production of the intermediate; and (ii) the mutant cell produces more of the metabolite than the parent cell when both cells are cultivated under the conditions; and (B) recovering the metabolite.
 52. A method of producing a first polypeptide, comprising(a) forming a mutant cell by introducing a nucleic acid construct into the genome of the parent cell, which comprises a first DNA sequence encoding the polypeptide, at a locus which is not within the first DNA sequence, a second DNA sequence encoding a protein that negatively regulates transcription, translation or secretion of a second polypeptide, and a third DNA sequence encoding a protease which hydrolyzes the polypeptide under conditions conducive to the production of the first polypeptide; (b) isolating the mutant cell which produces more of the polypeptide than the parent cell when both cells are cultivated under the conditions; (c) identifying the locus wherein the nucleic acid construct has been integrated; (d) producing a cell in which a corresponding locus has been disrupted; (e) culturing the cell under the conditions; and (f) recovering the first polypeptide.
 53. A method of producing a polypeptide, comprising(a) cultivating a mutant cell under conditions conducive for production of the polypeptide, wherein(i) the mutant cell is related to a parent cell, which comprises a first DNA sequence encoding the polypeptide, by the introduction of a nucleic acid construct into the genome of the parent cell at a locus which is not within the first DNA sequence and a second DNA sequence encoding a protease which hydrolyzes the polypeptide under the conditions, wherein the introduction of the nucleic acid construct specifically enhances transcription, translation or secretion of the polypeptide; and (ii) the mutant cell produces more of the polypeptide than the parent cell when both cells are cultivated under the conditions; and (b) recovering the polypeptide. 